Tocopherol-modified therapeutic drug compounds

ABSTRACT

Tocopherol-modified therapeutic drug compounds; emulsion, microemulsion, and micelle formulations that include the compounds; methods for making the compounds and formulations; methods for administering the compounds and formulations; and methods for treating conditions using the compounds and formulations.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.10/978,222, filed Nov. 29, 2004, which claims the benefit of U.S.Provisional Application No. 60/515,364, filed Oct. 29, 2003, U.S.Provisional Application No. 60/556,137, filed Mar. 24, 2004, U.S.Provisional Application No. 60/558,762, filed Apr. 1, 2004, and U.S.Provisional Application No. 60/621,655, filed Oct. 26, 2004, eachapplication is expressly incorporated by reference herein in itsentirety.

FIELD OF THE INVENTION

The present invention relates to new therapeutic drugs; compositionsthat include the new therapeutic drugs; and methods for administeringand using the new therapeutic drugs and compositions.

BACKGROUND OF THE INVENTION

The ability to administer biologically effective drugs that are poorlysoluble in biocompatible solvents to mammals has been a major hurdle inthe realm of pharmaceutical and medicinal chemistry. In particular,difficulties arise when an active drug is either insoluble in water orunstable in other biocompatible solvents.

One way to solubilize medicinal agents is to chemically modify them orconjugate them to another molecule to alter the solubility profile in aparticular solvent. Conjugates of active drugs, often referred to asprodrugs, include chemical derivatives of biologically-active parentcompounds that are converted into the parent compounds in vivo. Therelease of the active parent drug from the prodrug conjugate may occuras the result of processes such as hydrolysis or enzymatic cleavage. Therate of release is influenced by several factors, including the type ofchemical bond joining the active parent drug to the conjugate moiety.

Incorporating a water-soluble moiety (e.g., polyethylene glycol,polyglutamate, or polymer) to increase solubility and circulation lifeof a drug has been investigated by others. The use of fatty acids toconjugate to active drugs for purposes of tumor targeting has also beeninvestigated as a means of improving therapeutic index.

Many potent drugs, such as camptothecin and its analogues (e.g.,10-hydroxycamptothecin and 7-ethyl-10-hydroxycamptothecin), taxanes(e.g., paclitaxel, docetaxel), candesartan, amphotericin B,azathioprine, cyclosporine, entacapone, danazol, eletriptan, andbosentan, to name a few, are poorly soluble or have poor cellpermeability. Solubility problems of potential therapeutic agents arecommon and often cause delays in drug development. Several technologieshave been developed to facilitate the delivery of poorly soluble andinsoluble compounds to patients. Examples of technologies specificallydesigned to solve solubility problems include complexing agents,nanoparticles, microemulsions, solubility enhancing formulations,prodrugs, and novel polymer systems.

Paclitaxel (see structure below), a natural product found in the innerbark of the Pacific Yew tree, is an example of an importantchemotherapeutic agent with wide spectrum of activity against solidtumors, primarily breast, ovarian, colon and non-small cell lung cancer.

Paclitaxel exerts its antitumor activity by binding to tubulin andstabilizing microtubules and thus blocking cell mitosis. However,paclitaxel, like many other potent biologically active molecules, hasvery limited aqueous solubility.

Camptothecin (CPT) (see structure below) is another example of a poorlysoluble and difficult to formulate anti-cancer drug.

CPT is a quinoline-based alkaloid found in the bark of the Chinesecamptotheca tree and the Asian nothapodytes tree. CPT includes fourplanar rings (ABCD) and one boat conformational ring (E). CPT has beenfound to have a broad spectrum of antitumor activity, especially inhuman solid tumors. However, the lactone (ring E) of camptothecin andits derivatives is quite labile in alkaline condition and physiologicalpH. The opening of this ring to form an acid salt or carboxylate speciesresults in significant loss of anticancer activities. Efforts have beenmade since the early 1960s, when CPT was discovered by Wall and Wani, toimprove upon the anti-cancer activities of camptothecin and itsanalogues, and to reduce unwanted toxicities. No successful formulationof camptothecin has been developed to date because of its poorsolubility in both water and organic solvents. However, water-solubleanalogues of camptothecin, irinotecan hydrochloride (CAMPTOSAR) andtopotecan hydrochloride (HYCAMPTIN), have been developed and are theonly camptothecin analogs currently approved by the Food and DrugAdministration.

Recently, a vitamin E (α-tocopherol)-based emulsion formulationtechnology for paclitaxel drug delivery has been developed. In theformulation, paclitaxel is solubilized in α-tocopherol and formulated asan oil-in-water emulsion. However, while paclitaxel is soluble inα-tocopherol, the solubility of other active moieties (includingcamptothecin and other taxanes) in α-tocopherol is limited. Therefore,there continues to be a need for new methods, which are both safe andefficacious, of solubilizing and delivering poorly soluble active drugmolecules.

SUMMARY OF THE INVENTION

In one aspect, the present invention provides therapeutic drug compoundsthat have been modified to increase their lipophilicity. The compoundsof the invention include a one or more therapeutic drug moieties and oneor more lipophilic moieties. The therapeutic drug moiety is covalentlycoupled to the lipophilic moiety either directly or by a linker moiety.Methods for making the modified therapeutic drugs are also provided.

In another aspect of the invention, compositions that include thecompounds of the invention are provided. In one embodiment, thecomposition includes a compound of the invention, optionally one or moreother therapeutic agents, and a lipophilic medium. Methods for makingthe compositions are also provided.

In a further aspect, the invention provides emulsion and micelleformulations that include a compound of the invention. The emulsionformulation include an oil phase and an aqueous phase. The oil phaseincludes a compound of the invention and a lipophilic medium. Theemulsion may be an oil-in-water emulsion or a water-in-oil emulsion. Themicelle formulation includes a compound of the invention and an aqueousphase. Methods for making the emulsion and micelle formulations are alsoprovided.

In other aspects, methods for administering the compounds of theinvention to a subject in need thereof, and methods for treating acondition treatable by administration of a compound of the invention arealso provided.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing aspects and many of the attendant advantages of thisinvention will become more readily appreciated as the same become betterunderstood by reference to the following detailed description, whentaken in conjunction with the accompanying drawings, wherein:

FIG. 1 schematically illustrates reaction of d-60 -tocopherol and atherapeutic drug containing a carboxyl group to provide atocopherol-modified therapeutic drug compound;

FIG. 2 schematically illustrates tocopherol functionalization with acarbonyl chloride group (—C(═O)Cl) and a phosphoric chloride group(—P(═O)OR₁Cl), and reaction of the resulting acid chloride and anappropriately functionalized therapeutic drug compound to provide atocopherol-modified therapeutic drug compound;

FIG. 3 schematically illustrates tocopherol functionalization with adicarboxylic anhydride (succinic anhydride), and reaction of theresulting carboxylic acid and an appropriately functionalizedtherapeutic drug compound to provide a tocopherol-modified therapeuticdrug compound;

FIG. 4 schematically illustrates the preparation of tocopherol succinatecamptothecin;

FIG. 5 schematically illustrates the preparations of tocopherolsuccinate 10-hydroxycamptothecin and tocopherol succinate7-ethyl-10-hydroxycamptothecin;

FIG. 6 schematically illustrates the preparation of 10,20-di(tocopherolsuccinate) 7-ethyl-10-hydroxycamptothecin;

FIG. 7 schematically illustrates the preparation of tocopherol succinatecamptothecin containing a poly(ethylene oxide) group;

FIG. 8 schematically illustrates the preparation of tocopherol succinatepaclitaxel;

FIG. 9 schematically illustrates the preparation of tocopherol succinatedocetaxel;

FIG. 10 schematically illustrates the preparation of tocopherolterephthlate camptothecin;

FIG. 11 schematically illustrates the preparation of tocopherolcyclohexane-1,2-dicarboxylate 7-ethyl-10-hydroxycamptothecin;

FIG. 12 is a graph comparing the in vitro stability of the lactone formof camptothecin and of two representative tocopherol-modifiedtherapeutic drug compounds of the invention (SN2300, tocopherolsuccinate camptothecin; and SN2310, tocopherol succinate7-ethyl-10-hydroxycamptothecin);

FIG. 13 is a graph comparing GI₅₀ values reported by NCI forcamptothecin, irinotecan hydrochloride (irinotecan), and topotecanhydrochloride (topotecan) with GI₅₀ values obtained for tworepresentative tocopherol-modified therapeutic drug compounds of theinvention (SN2300 and SN2310) for cell lines: H460, HCT-116, HT29, andOVCAR-3;

FIGS. 14A and 14B are graphs of concentration-time values afterintravenous injection of 13.8 mg/kg of two representativetocopherol-modified therapeutic drug compounds of the invention (FIG.14A, SN2300; and FIG. 14B, SN2310); and

FIGS. 15A and 15B are graphs illustrating tumor growth (mm³) over timein xenographs treated with saline, irinotecan, and two representativetocopherol-modified therapeutic drug compounds of the invention (SN2300and SN2310) in two different tumor models (FIG. 15A, NCI-H460; and FIG.15B, HT-29).

DETAILED DESCRIPTION OF THE INVENTION

In one aspect, the present invention provides therapeutic drug compoundsthat have been modified to increase their lipophilicity. The compoundsof the invention are modified therapeutic drugs. The compounds of theinvention include a therapeutic drug moiety and a lipophilic moiety.

In some embodiments, the compounds of the invention include more thanone therapeutic drug moiety. In some embodiments, the compounds of theinvention include more than one lipophilic moiety. In other embodiments,the compounds of the invention include more than one therapeutic drugmoiety and more than one lipophilic moiety.

In some embodiments, the therapeutic drug moiety is covalently coupledto the lipophilic moiety through a linker moiety. In other embodiments,the therapeutic drug moiety is directly covalently coupled to thelipophilic moiety without a linker moiety.

In one embodiment, the lipophilic moiety is a tocopherol moiety, and thecompound is a tocopherol-modified therapeutic drug compound. Thetocopherol-modified therapeutic drug compound (or “tocopherolated”therapeutic drug compound) has one or more tocopherol moietiescovalently coupled to a therapeutic drug moiety or a tocopherol moietycovalently coupled to one or more therapeutic drug moieties. As notedabove, the tocopherol moiety is covalently coupled to the therapeuticdrug moiety either directly or through a linker moiety.

In one embodiment, the tocopherol-modified therapeutic drug compounds ofthe invention can be represented by the general formula (1):(T-L)_(n)(T)_(m)D  (1)wherein T is a tocopherol moiety (i.e., a representative lipophilicmoiety); L is a linker moiety; n is 0, 1, 2, or 3; m is 0, 1, 2, or 3;n+m is 1, 2, or 3; and D is a therapeutic drug moiety. In thisembodiment, the compound includes n T-L moieties (i.e.,tocopherol-linker moieties) and m tocopherol moieties, with the provisothat n+m is 1, 2, or 3. Each T-L moiety includes a tocopherol moietycovalently coupled to a linker moiety. Each of the n T-L moieties iscovalently coupled to the therapeutic drug moiety through the T-Lmoiety's linker moiety. In this embodiment, each of the m tocopherolmoieties is directly covalently coupled to the therapeutic drug moietywithout a linker moiety.

Representative compounds having formula (1) include those in which n is0 and m is 1, 2, or 3. These compounds have the general formula:T_(m)D  (2)In this embodiment, one, two, or three tocopherol moieties are directlycovalently coupled to the therapeutic drug moiety.

Representative compounds having formula (1) include those in which m is0 and n is 1, 2, or 3. These compounds have the general formula:(T-L)_(n)D  (3)In this embodiment, one, two, or three T-L moieties are covalentlycoupled to the therapeutic drug moiety through the T-L moiety's linkermoiety.

Representative compounds having formula (1) include those in which m is1 or 2, and n is 1 or 2. These compounds have the general formulae:(T-L)(T)D  (4)(T-L)(T)₂D  (5)(T-L)₂(T)D  (6)In these embodiments, the compounds of the invention have tocopherolmoieties that are directly covalently coupled to the therapeutic drugmoiety without a linker and tocopherol moieties that are covalentlycoupled to the therapeutic drug moiety through a linker (i.e., the T-Lmoieties).

The compounds of the invention described above include one therapeuticdrug moiety and one or more lipophilic moieties (e.g., tocopherolmoieties). In other embodiments, the compounds of the invention includemore than one therapeutic drug moiety. In one embodiment, the compoundsinclude two therapeutic drug moieties. In another embodiment, thecompounds include three therapeutic drug moieties. For compounds thatinclude more than one therapeutic drug moiety, the therapeutic drugmoieties a can be the same or different.

For compounds that include more than one therapeutic drug moiety, thetherapeutic drug moieties can be incorporated into the compound in anysuitable way. In some embodiments, the therapeutic drug moieties can bedirectly covalently coupled (e.g., the compound includes a -D-D- or -D-Dmoiety). In other embodiments, the therapeutic drug moieties areseparated in the compound by a linker moiety (e.g., the compoundincludes a -D-L-D- or -D-L-D moiety), a lipophilic moiety (e.g., thecompound includes a -D-T-D- or -D-T-D moiety), or a lipophilic-linkermoiety (e.g., the compound includes a -D-T-L-D-, -D-T-L-D, or -D-L-T-Dmoiety; or a -D-L-T-L-D- or -D-L-T-L-D moiety).

Representative compounds including two or three therapeutic drugmoieties have the general formula:(T-L)(D)_(p)  (7)wherein p is 2 or 3. In this embodiment, the two or three therapeuticdrug moieties are covalently coupled to the linker moiety. In thisinstance, the linker includes multiple sites for the attachment of thetherapeutic drug compound (or modified therapeutic drug compound). As isclear from formula (7), the linker moiety is also covalently coupled tothe lipophilic moiety (e.g., tocopherol moiety). As noted above,compounds of the invention including more than one therapeutic drugmoiety can have formulae other than shown above in formula (7). Forexample, such a compound can include more than one (e.g., two or three)lipophilic (e.g., tocopherol) moieties.

The compounds of the invention include one or more lipophilic moietiesand one or more therapeutic drug moieties that are either directlycovalently linked or covalently linked through linker moieties.

As used herein, the term “lipophilic moiety” refers to a chemical moietyhaving lipophilic or hydrophobic characteristics and that increases thesolubility of a therapeutic drug compound in a lipophilic solvent orenvironment when covalently coupled to the therapeutic drug compound toprovide a compound of the invention. A description of representativelipophilic moieties useful in making the compounds of the invention isprovided below.

As used herein, the term “therapeutic drug moiety” refers to a chemicalmoiety derived from a therapeutic drug compound. A description ofrepresentative therapeutic drug compounds useful in making the compoundsof the invention is provided below.

As used herein, the term “linker moiety” refers to an atom or a group ofatoms that covalently link, for example, a lipophilic moiety to atherapeutic drug moiety. A description of representative linkers usefulin making the compounds of the invention is provided below.

Lipophilic Modification of Therapeutic Drug Compounds.

A therapeutic drug compound may have one or more suitable functionalgroups, or may be modified to include one or more suitable functionalgroups for covalent coupling to a lipophilic moiety. Suitable functionalgroups include, for example, the following groups: hydroxyl group (—OH),amino group (primary amino group, —NH₂, or secondary amino group, —NHR₁,where R₁ is independently selected from H, C₁₋₆ n-alkyl, C₃₋₁₂ branchedalkyl, substituted or unsubstituted C₃₋₆ cycloalkyl, substituted orunsubstituted aryl, or aralkyl), thiol group (—SH), carboxyl group(—COOH), aldehyde group (—CHO), isocynato group (—N═C═O), sulfonic acidgroup (—SO₃H), sulfuric acid group (—OSO₃H), phosphoric acid group(—OPO₃H), phosphonic acid group (—PO₃H₂), allylic halide group, benzylichalide group, substituted benzylic halide group, and oxiranyl group(—CH(O)CH₂).

A therapeutic drug compound may be directly coupled to a lipophilicmoiety (e.g., a tocopherol moiety) through an ester group (—C(═O)O—),carbamate group (—OC(═O)NH—), sulfonate group (—SO₃—), sulfate group(—OSO₃—), phosphate group (—OPO₃R₁—, where R₁ is independently selectedfrom H, C₁₋₆ n-alkyl, C₃₋₁₂ branched alkyl, substituted or unsubstitutedC₃₋₆ cycloalkyl, substituted or unsubstituted aryl, or aralkyl),phosphonate group (—PO₃R₁—, where R₁ is independently selected from H,C₁₋₆ n-alkyl, C₃₋₁₂ branched alkyl, substituted or unsubstituted C₃₋₆cycloalkyl, substituted or unsubstituted aryl, or aralkyl), or ethergroup (—O—).

A tocopherol compound, a representative lipophilic compound suitable formaking the compounds of the invention, includes a hydroxyl group (—OH).After modification, a tocopherol compound may be covalently coupled to alinker compound that includes one or more reactive functional groups.Suitable reactive functional groups include the following groups:hydroxyl group (—OH), amino group (primary amino group, —NH₂, orsecondary amino group, —NHR₁, where R₁ is independently selected from H,C₁₋₆ n-alkyl, C₃₋₁₂ branched alkyl, substituted or unsubstituted C₃₋₆cycloalkyl, substituted or unsubstituted aryl, or aralkyl.), thiol group(—SH), carboxyl group (—C(═O)OH), aldehyde group (—CHO), isocynato group(—N═C═O), sulfonic acid group (—SO₃H), sulfuric acid group (—OSO₃H),phosphoric acid group (—OPO₃H), phosphonic acid group (—PO₃H₂), allylichalide group, benzylic halide group, substituted benzylic halide group,and oxiranyl group (—CH(O)CH₂).

Linker Moieties.

As noted above, in some embodiments, the compounds of the inventioninclude a lipophilic moiety (e.g., tocopherol moiety) covalently coupledto a therapeutic drug moiety by a linker moiety. In addition to theembodiments described above, the tocopherol-modified therapeutic drugcompounds of the invention can be represented by the general formula(8):T-A-R-A′-D  (8)where T is a tocopherol moiety (i.e., a representative lipophilicmoiety), D is a therapeutic drug moiety, and A-R-A′ is a linker moiety.It will be appreciated that for formulae (1) and (3)-(7) above, each ofwhich includes linker moiety L, the linker moiety L in those compoundscan be linker moiety A-R-A′.

In formula 8, groups A and A′ are independently selected from O, S, SO,SO₂, NR₁, carboxylate group (—C(═O)O—), amide group (—C(═O)NR₁—),anhydride group (—C(═O)OC(═O)—), carbamate group (—OC(═O)NH—),carbonyldioxy group (—OC(═O)O—), ureylene group (—NR₁C(═O)NR₂—),phosphate group (—OP(═O)(OR₁)O—), phosphamide group (—OP(═O)(NR₁)O—),phosphonate group (—OP(OR₁)O—), phosphonamide group(—OP(═O)NR₁—),sulfate group (—OSO₂—), sulfamide group (—SO₂NR₁—), sulfonate group(—SO₃—), sulfonamide group (—SO₂NR₁—), and the dicarbonyl group,—C(═O)R₃C(═O)—, where R₃ is absent or a divalent alkyl (e.g.,—(CH₂)_(n)—, n=1-12), substituted alkyl, branched alkyl, cycloalkyl,substituted cycloalkyl, aryl, substituted aryl. For the above groups, Ris a divalent group selected from the following groups: alkyl;substituted alkyl, branched alkyl; cycloalkyl; substituted cycloalkyl;heteroalkyl; substituted heteroalkyl; aryl; substituted aryl; aralkyl;substituted aralkyl; amino acid; peptide; polypeptide; protein; mono-,di- or polysaccharide; oligomer of ethylene glycol, poly(ethyleneglycol); poly(alkylene oxide) polymers, such as poly(ethylene oxide) andpoly(propylene oxide); and poly(ethylene oxide)-poly(propylene oxide)copolymer. For the above groups, R₁ and R₂ are independently selectedfrom Na⁺, K⁺, H, C₁₋₆ n-alkyl, C₃₋₁₂ branched alkyl, substituted orunsubstituted C₃₋₆ cycloalkyl, substituted or unsubstituted aryl, andsubstituted or unsubstituted aralkyl.

As used herein, the term “aryl” refers to monocyclic and polycyclicaromatic compounds having from 6 to 14 carbon or hetero atoms, andincludes carbocyclic aryl groups and heterocyclic aryl groups.Representative aryl groups include phenyl, naphthyl, pyridinyl,pyrimidinyl, thiazolyl, indolyl, imidazolyl, furanyl, and the like. Theterm “aralkyl” refers to an alkyl group that is substituted with an arylgroup.

The following compounds (compounds 9 to 32) are representative examplesof compounds having formula 8.

A linker moiety (L) and therapeutic drug moiety (D) may be covalentlycoupled through an ester group. In one embodiment, the therapeutic drugmoiety includes a hydroxyl group that is coupled with a carboxyl groupof the linker moiety. The linker moiety may be coupled to a tocopherolmoiety through an ether group (compound 9), ester group (compound 10),amine group (compound 11), or amide group (compound 12).

In another embodiment, the therapeutic drug moiety (D) includes acarboxyl group that is coupled with a hydroxyl group of the linkermoiety (L). The linker moiety may be coupled to a tocopherol moietythrough an ether group (compound 13), ester group (compound 14), aminegroup (compound 15), or amide group (compound 16).

In the above compounds, divalent group R is selected from alkyl;substituted alkyl; branched alkyl; cycloalkyl; substituted cycloalkyl;heteroalkyl; substituted heteroalkyl; aryl; substituted aryl; aralkyl;substituted aralkyl; amino acid; peptide; polypeptide; protein; mono-,di- or polysaccharide; oligomer of ethylene glycol, poly(ethyleneglycol); poly(alkylene oxide) polymers, such as poly(ethylene oxide) andpoly(propylene oxide); and poly(ethylene oxide)-poly(propylene oxide)copolymer. In the above compounds, R₁ is selected from H, C₁₋₆ n-alkyl,C₃₋₁₂ branched alkyl, substituted or unsubstituted C₃₋₆ cycloalkyl,substituted or unsubstituted aryl, and substituted or unsubstitutedaralkyl.

While the preferred embodiment of the invention has been illustrated anddescribed, it will be appreciated that various changes can be madetherein without departing from the spirit and scope of the invention. Alinker moiety (L) and therapeutic drug moiety (D) may be covalentlycoupled through an amide group. In one embodiment, the therapeutic drugmoiety includes an amine group that is coupled a carboxyl group of thelinker moiety. The linker moiety may be coupled to a tocopherol moietythrough an ether group (compound 17), ester group (compound 18), aminegroup (compound 19), or amide group (compound 20).

In the above compounds, divalent group R is selected from alkyl;substituted alkyl; branched alkyl; cycloalkyl; substituted cycloalkyl;heteroalkyl; substituted heteroalkyl; aryl; substituted aryl; aralkyl;substituted aralkyl; amino acid; peptide; polypeptide; protein; mono-,di- or polysaccharide; oligomer of ethylene glycol, poly(ethyleneglycol); poly(alkylene oxide) polymers, such as poly(ethylene oxide) andpoly(propylene oxide); and poly(ethylene oxide)-poly(propylene oxide)copolymer. In the above compounds, R₁ and R₂ are independently selectedfrom H, C₁₋₆ n-alkyl, C₃₋₁₂ branched alkyl, substituted or unsubstitutedC₃₋₆ cycloalkyl, substituted or unsubstituted aryl, and substituted orunsubstituted aralkyl.

A linker moiety (L) and a therapeutic drug moiety (D) may be covalentlycoupled through an ether group (compound 21) or amine group (compound22). In one embodiment, the therapeutic drug moiety includes a hydroxygroup, and in another embodiment, the therapeutic drug moiety includesan amine group. The linker moiety may be coupled to a tocopherol moietythrough an ether group.

In the above compounds, R₁ is selected from H, C₁₋₆ n-alkyl, C₃₋₁₂branched alkyl, substituted or unsubstituted C₃₋₆ cycloalkyl,substituted or unsubstituted aryl, or aralkyl.

A tocopherol moiety (T) and a therapeutic drug moiety (D) may becovalently coupled through a carbonyldioxy group (—OC(═O)O—) (compound23). In this case, the linker moiety is the carbonyldioxy group and thetherapeutic drug moiety includes a hydroxyl group.

A tocopherol moiety (T) and a therapeutic drug moiety (D) may becovalently coupled through an anhydride group (—C(═O)OC(═O)—). In oneembodiment, the therapeutic drug moiety includes a carboxyl group thatis coupled with a carboxyl group of the linker moiety. The linker moietymay be coupled to a tocopherol moiety through an ether group (compound24), ester group (compound 25), amine group (compound 26), or amidegroup (compound 27).

In the above compounds, divalent group R is selected from alkyl;substituted alkyl; branched alkyl; cycloalkyl; substituted cycloalkyl;heteroalkyl; substituted heteroalkyl; aryl; substituted aryl; aralkyl;substituted aralkyl; amino acid; peptide; polypeptide; protein; mono-,di- or polysaccharide; oligomer of ethylene glycol, poly(ethyleneglycol); poly(alkylene oxide) polymers, such as poly(ethylene oxide) andpoly(propylene oxide); and poly(ethylene oxide)-poly(propylene oxide)copolymer. In the above compounds, R₁ is selected from H, C₁₋₆ n-alkyl,C₃₋₁₂ branched alkyl, substituted or unsubstituted C₃₋₆ cycloalkyl,substituted or unsubstituted aryl, and substituted or unsubstitutedaralkyl.

A tocopherol moiety (T) and therapeutic drug moiety (D) may becovalently coupled through a phosphate, phosphoramide, or thiophosphategroup (compound 28).

In the above compounds, X is O, NR₂, or S; X₁ is O, NR₃, or S; and R₁ isselected from Na⁺, K⁺, H, C₁₋₆ n-alkyl, C₃₋₁₂ branched alkyl,substituted or unsubstituted C₃₋₆ cycloalkyl, substituted orunsubstituted aryl, or aralkyl; and R₂ and R₃ are independently selectedfrom C₁₋₆ n-alkyl, C₃₋₁₂ branched alkyl, substituted or unsubstitutedC₃₋₆ cycloalkyl, substituted or unsubstituted aryl, or aralkyl.

A tocopherol moiety (T) and a therapeutic drug moiety (D) may becovalently coupled through a sulfate, thiosulfate, or sulfonamide group(compound 29).

In the above compounds, X is O, NR₁, or S; and R₁ is selected from H,C₁₋₆ n-alkyl, C₃₋₁₂ branched alkyl, substituted or unsubstituted C₃₋₆cycloalkyl, substituted or unsubstituted aryl, or aralkyl.

A tocopherol moiety (T) and therapeutic drug moiety (D) may becovalently coupled through a ureylene group (—NHC(═O)NH—) (compound 30).

In the above compounds, R₁ and R₂ are independently selected from H,C₁₋₆n-alkyl, C₃₋₁₂ branched alkyl, substituted or unsubstituted C₃₋₆cycloalkyl, substituted or unsubstituted aryl, or aralkyl.

A tocopherol moiety (T) and a therapeutic drug moiety (D) may becovalently coupled through a carbamate group (—NR₁C(═O)O— or—OC(═O)NR₂—, compounds 31 and 32, respectively).

In the above compounds, R₁ and R₂ are independently selected from H,C₁₋₆n-alkyl, C₃₋₁₂ branched alkyl, substituted or unsubstituted C₃₋₆cycloalkyl, substituted or unsubstituted aryl, or aralkyl.

Lipophilic Moiety.

The compounds of the invention include one or more lipophilic moieties.The lipophilic moiety or moieties increases the solubility of thecompound in a lipophilic solvent or environment. In one embodiment, thelipophilic moiety is a tocopherol moiety.

As used herein, the term “tocopherol moiety” refers to a chemical moietythat is derived from a family of natural or synthetic compounds, alsoknown by their generic names, tocol or vitamin E. In addition totocopherol compounds, tocotrienol compounds are included in this family.These compounds include a chroman head having a phenolic alcohol (C-6)and a phytyl tail (C-2). These compounds have the following generalformula:

Tocopherols constitute a series of related benzopyranols (or methyltocols) in which the C-2 phytyl (sixteen carbon) side chain issaturated. Representative tocopherols include α-tocopherol, (d-form,dl-form, l-form), β-tocopherol (d-form, dl-form, l-form), γ-tocopherol(d-form, dl-form, l-form), and δ-tocopherol (d-from, dl-form, l-form).Among tocopherols, α-tocopherol is the most abundant. Tocotrienols aresimilar in structure to tocopherols except that the trienols have threedouble bonds in the C-2 phytyl side chain.

Tocopherol and tocotrienol compounds useful in making the compounds ofthe invention include those shown below.

Compound R₁ R₂ R₃ alpha (α) CH₃ CH₃ CH₃ beta (β) CH₃ H CH₃ gamma (γ) HCH₃ CH₃ delta (δ) H H CH₃As used herein, the term “tocopherol” refers to any member of thetocopherol family noted above.

Therapeutic Drug Moiety.

The compounds of the invention include one or more therapeutic drugmoieties. Virtually any therapeutic drug compound having a suitablefunctional group, or that can be modified to include a suitablefunctional group, can be covalently coupled to a lipophilic compound toprovide a compound of the invention. Representative functional groupsinclude, for example, hydroxyl groups (—OH), amino groups (primary aminogroups, —NH₂, and secondary amino groups, —NHR), thiol groups (—SH),carboxyl groups (—COOH), aldehyde groups (—CHO), isocynato groups(—N═C═O), sulfonic acid groups (—SO₃H), sulfuric acid groups (—OSO₃H),phosphoric acid groups (—OPO₃H), phosphonate groups (—PO₃OR₁R₂, and R₁and R₂ are independently selected from H, C₁₋₆ n-alkyl, C₃₋₁₂ branchedalkyl, substituted or unsubstituted C₃₋₆ cycloalkyl, substituted orunsubstituted aryl, or aralkyl.), allylic halide group, benzylic halidegroup, substituted benzylic halide group, and oxiranyl group(—CH(O)CH₂).

Therapeutic drug compounds useful in making the compounds of theinvention need not be substantially water insoluble, although tocopherolmodification according to the present invention is especially wellsuited for formulating and delivering such water-insoluble compounds.

In one embodiment, the therapeutic drug moiety is derived from atherapeutic compound that is substantially insoluble in water. Inanother embodiment, the therapeutic drug moiety is derived from atherapeutic compound that is substantially insoluble in organicsolvents. In another embodiment, the therapeutic drug moiety is derivedfrom a therapeutic compound that is substantially insoluble in water andsubstantially insoluble in organic solvents. In one embodiment, thetherapeutic drug compound has a solubility in water at room temperatureless than about 1000 μg/mL. In one embodiment, the therapeutic drugcompound has a solubility in water at room temperature less than about500 μg/mL. In one embodiment, the therapeutic drug compound has asolubility in water at room temperature less than about 100 μg/mL. Inone embodiment, the therapeutic drug compound has a solubility in waterat room temperature less than about 25 μg/mL.

Representative therapeutic compound drugs useful in making the compoundsof the invention include anticancer compounds (e.g., paclitaxel and itsderivatives including docetaxel, camptothecin and its derivativesincluding 7-ethyl-10-hydroxycamptothecin (SN38) and10-hydroxycamptothecin, and doxorubicin and its derivatives), antifungalcompounds (e.g., flucanazole), antibacterial compounds (e.g., penicillinG, penicillin V), anti-hypertensive compounds (e.g., hydralazine,candesartan, and carvediol), anti-inflammatory compounds (e.g.,isoxicam), antidiabetic compounds (e.g., metformin), antiviral compounds(e.g., lamivudine), antidepressant compounds (e.g., fluoxetine),antihistaminic compounds (e.g., hydroxyzine), anti-arrhythmic compounds(e.g., procainamide hydrochloride), anti-hyperlipoproteinemic compounds(e.g., probucol), and compounds for reproductive health (e.g., danazol),and treating Parkinson's disease (e.g., lazabemide), andimmunosuppressive (e.g., azathioprine and cyclosporine) and respiratory(e.g., bosentan) diseases and conditions. Other therapeutically usefulbiological materials that can be modified according to the invention,include biologically active proteins, enzymes, and peptides.

In one embodiment, the therapeutic drug moiety is derived from ananticancer compound. Representative anticancer therapeutic compoundsinclude taxanes. Taxanes include any anti-mitotic taxane, taxanederivative or analog. As used herein, the term “taxane” refers totaxanes, taxines, and taxoids, as well as derivatives or analogsthereof.

Paclitaxel and its derivatives and analogs are members of the taxanefamily. Paclitaxel derivatives include, for example benzoate derivativesof paclitaxel such as 2-debenzoyl-2-aroyl and C-2-acetoxy-C-4-benzoatepaclitaxel, 7-deoxytaxol, C-4 aziridine paclitaxel, as well as variouspaclitaxel conjugates with natural and synthetic polymers, particularlywith fatty acids, phospholipids, and glycerides and1,2-diacyloxypropane-3-amine. Other paclitaxel derivatives includedocetaxel; spicatin; taxane-2,13-dione, 5β,9β,10β-trihydroxy-, cyclic9,10-acetal with acetone, acetate; taxane-2,13-dione,5β,9β,10β-trihydroxy-, cyclic 9,10-acetal with acetone;taxane-2β,5β,9β,10β-tetrol, cyclic 9,10-acetal with acetone; taxane;cephalomannine-7-xyloside; 7-epi-10-deacetylcephalomannine;10-deacetylcephalomannine; cephalomannine; taxol B;13-(2′,3′-dihydroxy-3′phenylpropionyl)baccatin III; yunnanxol;7-(4-azidobenzoyl)baccatin III; N-debenzoyltaxol A; O-acetylbaccatin IV;7-(triethylsilyl)baccatin III;7,10-di-O-[(2,2,2,-trichloroethoxy)carbonyl]baccatin III; baccatin III13-O-acetate; baccatin diacetate; baccatin; baccatin VII; baccatin VI;baccatin IV; 7-epi-baccatin III; baccatin V; baccatin I; baccatin III;baccatin A; 10-deactyl-7-epitaxol; epitaxol; 10-deacetyltaxol C;7-xylosyl-10-deacetyltaxol; 10-deacetyltaxol-7-xyloside;7-epi-10-deacetyltaxol; 10-deactyltaxol; or 10-deactyltaxol B.

Other anticancer compounds useful in making the compounds of theinvention include camptothecin and its derivatives including7-ethyl-10-hydroxycamptothecin (SN38) and 10-hydroxycamptothecin, anddoxorubicin and its derivatives.

In certain embodiments, the therapeutic drug moiety is derived frompaclitaxel, docetaxel, camptothecin, or their derivatives.

For compounds of the invention having formula (2) with m=1, formula (3)with n=1, and formula 8, certain compounds are excluded and are notwithin the scope of the invention. When the linker moiety is2-hydroxypropylene (—CH₂CH(OH)CH₂—), the therapeutic drug moiety is notan α-amino acid (e.g., glycine, alanine, proline, cysteine, aminobutyricacid, aspartic acid, glutamic acid), an ω-amino acid (e.g., β-alanine,γ-aminobutyric acid, ε-aminocaproic acid, 2-aminoethanesulfonic acid(taurine)), or a peptide containing a cysteine residue bonding throughits N-terminus or thiol group (e.g., -glutathione). When the linkermoiety is a succinate, the therapeutic drug moiety is not an S-linkedamino or amino acid compound coupled to one of the aliphatic succinatecarbons. When the linker moiety is succinate, the therapeutic drugmoiety is not ferulic acid or an ester thereof.

In another aspect, methods for making the compounds of the invention areprovided. There are many ways to covalently couple a lipophilic compound(e.g., a tocopherol compound) to a therapeutic drug compound to form acompound of the invention. In one embodiment, a representativetocopherol, d-α-tocopherol, includes a hydroxyl group that may bedirectly coupled with a carboxyl group of a therapeutic drug to form atocopherol-modified therapeutic drug compound. The preparation of arepresentative tocopherol-modified therapeutic compound of the inventionfrom a carboxylic acid-containing therapeutic drug compound isillustrated in FIG. 1.

In another embodiment, a tocopherol may be functionalized at thehydroxyl group with a reagent to attach an active group such asphosphoric chloride (—P(O)OR₁Cl), phosphonic chloride (—P(O)R₁Cl),sulfonic chloride (—SO₂Cl), or carbonyl chloride (—COCl). The resultingacid chloride can then be reacted with an appropriately functionalizedtherapeutic drug compound to provide a tocopherol-modified therapeuticdrug compound.

In FIG. 2, X is O, S, or NH; and R₁ is independently selected from H,C₁₋₆ n-alkyl, C₃₋₁₂ branched alkyl, substituted or unsubstituted C₃₋₆cycloalkyl, substituted or unsubstituted aryl, and substituted orunsubstituted aralkyl.

In another embodiment, a tocopherol may be functionalized at thehydroxyl group with a dicarboxylic acid, ester, or anhydride reagent.Suitable reagents include succinic acid anhydride,1,2-cyclohexanedicarboxylic anhydride, 2,3-dimethylsuccinic anhydride,3,3-tetramethylene glutaric anhydride, glutaric anhydride, maleic acidanhydride, phthalic acid anhydride, terephthalic acid, or isophthalicacid to attach a carboxyl group (—COOH). The resulting carboxyl groupmay then be directly reacted with an appropriately functionalizedtherapeutic drug or the carboxyl group may be converted to a morereactive carbonyl chloride group (—COCl), and then the carbonyl chloridegroup may be coupled with the functional group of the therapeutic drugto form a tocopherol-modified therapeutic drug compound as illustratedin FIG. 3. In FIG. 3, X is O, S, NH, or C(═O)O.

In another embodiment, a linker can be coupled to the hydroxyl group ofa tocopherol and then a therapeutic drug can be coupled to an accessiblefunctional group on the linker. The functional group may be, forexample, but not limited to, a carboxyl group (—COOH), a poly(ethyleneoxide) group (—(CH₂CH₂O)_(n)—H), an aldehyde group (—CHO), an isocyanatogroup (—N═C═O), a phosphoric acid group (—OPO₃H₂), or phosphoricchloride group (—OPO₂R₁Cl, where R₁ is a substituted or unsubstitutedalkyl or cycloalkyl, a substituted or unsubstituted aryl, or anaralkyl), a phosphonic chloride group (—PO₂R₁Cl, where R₁ is asubstituted or unsubstituted alkyl or cycloalkyl, a substituted orunsubstituted aryl, or an aralkyl), a sulfuric acid group (—OSO₃H₂), achlorosulfuric group (—SO₃Cl), or an oxiranyl group (—CH(O)CH₂).

The syntheses of representative tocopherol-modified therapeutic drugcompounds of the invention are illustrated in FIGS. 4-11 and describedin Examples 1-13.

FIG. 4 illustrates the preparation of a tocopherol succinatecamptothecin compound. Tocopherol succinic acid (vitamin E succinicacid) has a free carboxyl group that can couple with a hydroxyl group,amino group, thiol group, or carbonyl chloride group to provide atocopherol-modified therapeutic drug having a succinate group as alinker. In FIG. 4, the carboxyl group of tocopherol acid succinate iscoupled with the hydroxyl group of camptothecin. The preparation oftocopherol succinate camptothecin is described in Example 1.

FIG. 5 illustrates the preparation of tocopherol succinate10-hydroxycamptothecin and tocopherol succinate7-ethyl-10-hydroxycamptothecin (SN38). Tocopherol succinic acid isconverted to the corresponding acid chloride, and then reacted with10-hydroxycamptothecin or 7-ethyl-10-hydroxycamptothecin (SN38). Thepreparations of tocopherol succinate 10-hydroxycamptothecin andtocopherol succinate 7-ethyl-10-hydroxycamptothecin are described inExamples 2 and 3, respectively.

FIG. 6 illustrates the preparation of 10,20-di(tocopherol succinate)SN38, which contains one therapeutic drug (SN38) moiety, two tocopherolmoieties, and two linker moieties (succinyl groups). The preparation of10,20-di(tocopherol succinate) SN38 is described in Example 4.

Suitable linker moieties can include an oligomer or polymer such as apeptide, polypeptide, protein, mono-, di- or polysaccharide, oligomer ofethylene glycol, poly(ethylene glycol), poly(alkylene oxide) such aspoly(ethylene oxide) and poly(propylene oxide), or poly(ethyleneoxide)-poly(propylene oxide) copolymer. FIG. 7 illustrates thepreparation of a tocopherol-modified camptothecin containing a linkermoiety that includes a poly(ethylene oxide) group. The preparation ofthe tocopherol succinate camptothecin having a linker moiety thatincludes a poly(ethylene oxide) group is described in Example 5.

FIG. 8 illustrates the preparation of tocopherol succinate paclitaxel.In the preparation, tocopherol succinic acid is converted to thecorresponding acid chloride and then reacted with paclitaxel. Thepreparation of tocopherol succinate paclitaxel is described in Example6. FIG. 9 illustrates the preparation of tocopherol succinate docetaxel.The preparation of tocopherol succinate docetaxel is described inExample 7.

FIG. 10 illustrates the preparation of tocopherol terephthalatecamptothecin. In the preparation, tocopherol is first conjugated withterephthalate to form tocopherol terephthalate (Example 9) and is thencoupled with camptothecin to form tocopherol terephthalate camptothecin.The preparation of tocopherol terephthalate camptothecin is described inExample 10.

FIG. 11 illustrates the preparation of tocopherolcyclohexane-1,2-dicarboxylate SN38. The preparation of tocopherolcyclohexane-1,2-dicarboxylate SN38 is described in Example 11.

The preparations of tocopherol succinate doxorubicin and tocopherolsuccinate hydroxyzine are described in Examples 12 and 13, respectively.

In another aspect, the present invention provides compositions thatinclude the compounds of the invention. The compositions include one ormore compounds of the invention, optionally one or more additionaltherapeutic agents, and a lipophilic medium. In one embodiment, atocopherol-modified therapeutic drug compound is dissolved in thelipophilic medium. Because of the lipophilic moiety, the compound hasimproved lipophilicity compared to the unmodified therapeutic drugcompound. The lipophilic medium (or carrier) of the composition can beany one of a variety of lipophilic mediums including, for example, oils.In one embodiment, the lipophilic medium includes a tocopherol (e.g.,α-tocopherol). Representative oils useful as the lipophilic mediuminclude the following:

Fatty acids and esters thereof, including carboxylic acids of variouschain lengths, mostly straight chain, but which could be branched,examples of which include capric, caprylic, caproic, lauric, myristic,stearic, oleic, linoleic, behenic, and as well as saturated orunsaturated fatty acids and esters;

Fatty acids esterified with glycerin to form mono-, di-, ortriglycerides, which can be synthetic or derived from natural sources,including, but not limited to, for example, glycerides such as soybeanoil, cottonseed oil, rapeseed oil, fish oil, castor oil, Capmul MCM,Captex 300, Miglyol 812, glyceryl monooleate, triacetin, acetylatedmonoglyceride, tristearin, glyceryl behenate, and diacetyl tartaric acidesters of monoglycerides;

Glycerides conjugated to other moieties, such as polyethylene glycol(for example, Labrasol, Labrafac, Cremophor EL);

Phospholipids, either natural or synthetic, such as dimyristylphosphatidylcholine, egg lecithin, and pegylated phospholipids;

Other fatty esters including fatty alcohols (myristyl myristate,isopropyl palmitate), or sugars (sorbitan monooleate, SPAN 80, Tween 80,sucrose laurate);

Fatty alcohols such as stearyl alcohol, lauryl alcohol, benzyl alcohol,or esters or ethers thereof, such as benzyl benzoate;

Fat-soluble vitamins and derivatives, for example, vitamin E (includingall of the tocopherols and tocotrienols, and tocopherol and tocotrienolderivatives, such as vitamin E succinate, vitamin E acetate, and vitaminE succinate polyethylene glycol (TPGS)).

Organic co-solvents can also be used in the compositions, optionally incombination with water, including for example, ethanol, polyethyleneglycol, propylene glycol, glycerol, N-methyl pyrrolidone, and dimethylsulfoxide.

The solubilities of two representative tocopherol-modified camptothecincompounds of the invention in several mediums are compared tocamptothecin in Example 14.

In a further aspect, the invention provides emulsion, microemulsion, andmicelle formulations that include a compound of the invention. Methodsfor making the emulsion, microemulsion, and micelle formulations arealso provided.

As used herein, the term “emulsion” refers to a colloidal dispersion oftwo immiscible liquids, such as an oil and water, in the form ofdroplets, whose diameter, in general, are between 0.1 and 3.0 micronsand which is typically optically opaque, unless the dispersed andcontinuous phases are refractive index matched. Such systems possess afinite stability, generally defined by the application or relevantreference system, which may be enhanced by the addition of amphiphilicmolecules or viscosity enhancers.

The term “microemulsion” refers to a thermodynamically stableisotropically clear dispersion of two immiscible liquids, such as an oiland water, stabilized by an interfacial film of surfactant molecules.The microemulsion has a mean droplet diameter of less than 200 nm, ingeneral between 10-50 nm. In the absence of water, mixtures of oil(s)and non-ionic surfactant(s) form clear and isotropic solutions that areknown as self-emulsifying drug delivery systems (SEDDS) and can be usedto improve lipophilic drug dissolution and oral absorption.

The emulsion and microemulsion formulations include an oil phase and anaqueous phase. The emulsion or microemulsion can be an oil-in-wateremulsion or a water-in-oil emulsion. The oil phase includes one or morecompounds of the invention and a lipophilic medium, as described above.In one embodiment, the compound is present in the formulation in anamount from about 0.005 to about 3.0 weight percent based on the totalweight of the formulation. In one embodiment, the compound is present inthe formulation in an amount from about 0.01 to about 2.5 weight percentbased on the total weight of the formulation. In one embodiment, thecompound is present in the formulation in an amount from about 0.1 toabout 1.5 weight percent based on the total weight of the formulation.In one embodiment, the lipophilic medium is present in the formulationin an amount from about 2 to about 20 weight percent based on the totalweight of the formulation. In one embodiment, the lipophilic medium ispresent in the formulation in an amount from about 4 to about 12 weightpercent based on the total weight of the formulation. In one embodiment,the lipophilic medium is present in the formulation in an amount fromabout 6 to about 10 weight percent based on the total weight of theformulation.

In one embodiment of the emulsion or microemulsion, the compound is atocopherol-modified therapeutic drug compound, the lipophilic mediumincludes a tocopherol, and the aqueous medium is water.

In addition to the compounds of the invention, the emulsion ormicroemulsion formulations can include other components commonly used inemulsions an microemulsions, and particularly used in pharmaceuticalemulsions and microemulsions. These components include surfactants andco-solvents, among others. Representative surfactants include nonionicsurfactants such as surface active tocopherol derivatives and surfaceactive polymers.

Suitable surface active tocopherol derivatives include tocopherolpolyethylene glycol derivatives, such as vitamin E succinatepolyethylene glycol (e.g., d-α-tocopherol polyethylene glycol 1000succinate, TPGS), which is a vitamin E derivative in which apolyethylene glycol is attached by a succinic acid ester at the ringhydroxyl of vitamin E. As used herein, “vitamin E succinate polyethyleneglycol” includes vitamin E succinate polyethylene glycol and derivativesof vitamin E polyethylene glycol having various ester and ether links.TPGS is a non-ionic surfactant (HLB=16-18). TPGS is reported to inhibitP-glycoprotein, a protein that contributes to the development ofmulti-drug resistance. Embodiments of the formulations of the inventionthat include TPGS therefore include a P-glycoprotein inhibitor. Surfaceactive tocopherol derivatives (e.g., TPGS) can be present in theformulations of the invention in an amount from about 1 to about 10weight percent, about 2 to about 6 weight percent, or about 5 weightpercent, based on the total weight of the formulation.

Suitable nonionic surfactants include block copolymers of ethylene oxideand propylene oxide known as POLOXAMERS or PLUROINICS. These syntheticblock copolymers of having the general structure:H(OCH₂CH₂)_(a)(OC₃H₆CH₂)_(b)(OCH₂CH₂)_(a)OH. The following variantsbased on the values of a and b are commercially available from BASFPerformance Chemicals (Parsippany, N.J.) under the trade name PLURONICand consist of the group of surfactants designated by the CTFA name ofPOLOXAMER 108, 188, 217, 237, 238, 288, 338, 407, 101, 105, 122, 123,124, 181, 182, 183, 184, 212, 231, 282, 331,401, 402, 185, 215, 234,235, 284, 333, 334, 335, and 403. For the most commonly used POLOXAMERS124, 188, 237, 338, and 407 the values of a and b are 12/20, 79/28,64/37, 141/44 and 101/56, respectively. In one embodiment the nonionicsurfactant is present in the formulation in an amount from about 0.5 toabout 5 weight percent based on the total weight of the formulation.

Co-solvents useful in the formulations include ethanol, polyethyleneglycol, propylene glycol, glycerol, N-methylpyrrolidone, dimethylamide,and dimethylsulfoxide, among others. Polyethylene glycol (PEG) is ahydrophilic, polymerized form of ethylene glycol, consisting ofrepeating units having the chemical structure: (—CH₂CH₂O—). The generalformula for polyethylene glycol is H(OCH₂CH₂)_(n)OH. The molecularweight ranges from 200 to 10,000. Such various forms are described bytheir molecular weights, for example, PEG-200, PEG-300, PEG-400, and thelike.

Paclitaxel emulsions and their components are described in U.S. Pat. No.6,458,173 and U.S. Pat. No 6,660,286, each expressly incorporated hereinby reference in its entirety.

Representative emulsions including tocopherol-modified therapeutic drugcompounds (e.g., tocopherol succinate docetaxel, tocopherol succinatepaclitaxel, tocopherol succinate camptothecin, tocopherol succinate7-ethyl-10-hydroxycamptothecin, and tocopherol succinate10-hydroxycamptothecin) are described in Example 15. In vitrocytotoxicities of representative tocopherol-modified therapeutic drugcompounds (e.g., tocopherol succinate 7-ethyl-10-hydroxycamptothecin andtocopherol succinate camptothecin) are described in Example 16.

In a further aspect, the invention provides micelle formulations thatinclude a compound of the invention and an aqueous phase. Micelles areorganized aggregates of one or more surfactants in solution. In oneembodiment, the compound is present in the formulation in an amount fromabout 0.005 to about 3.0 weight percent based on the total weight of theformulation. In one embodiment, the compound is present in theformulation in an amount from about 0.01 to about 2.5 weight percentbased on the total weight of the formulation. In one embodiment, thecompound is present in the formulation in an amount from about 0.1 toabout 1.0 weight percent based on the total weight of the formulation.Suitable surfactants include those noted above, and in the amounts notedabove. In one embodiment of the micelle formulation, the compound is atocopherol-modified therapeutic drug compound and the surfactant istocopherol polyethylene glycol succinate (TPGS). Representative micelleformulations including tocopherol-modified therapeutic drug compoundsare described in Example 15.

The micelle formulation can also include additional components such asco-solvents including those noted above. In one embodiment, the micelleformulation includes a polyethylene glycol and a lower alkyl alcohol(e.g., ethanol). In one embodiment, the co-solvents are present in anamount from about 2 to about 20 weight percent based on the total weightof the formulation. The micelle, emulsion, and microemulsionformulations include an aqueous phase. In one embodiment, the aqueousphase includes deionized water. In another embodiment, the aqueous phaseincludes saline. In another embodiment, the aqueous phase is salinebuffered with an organic acid (e.g., succinate, citrate).

The invention also provides the use of the compounds of the invention inthe manufacture of a medicament. For example, for compounds of theinvention that include a therapeutic drug moiety derived from atherapeutic drug compound effective in treating cell proliferativedisease, the invention provides the use of such compounds in themanufacture of a medicament for the treatment of cell proliferativedisease.

In other aspects, methods for administering a compound of the inventionto a subject in need thereof, and methods for treating a conditiontreatable by administration of a therapeutically effective amount of acompound of the invention are also provided. These methods include theadministration of the compounds, compositions, emulsion formulations,microemulsion formulations, and micelle formulations described herein.

In one embodiment, the invention provides a method for treating acondition that is treatable by the parent, unmodified therapeutic drugcompound (e.g., a cell proliferative disease such as cancer). In themethod, a therapeutically effective amount of a compound of theinvention is administered to a subject in need thereof.

In one embodiment, the invention provides a method for treating a cellproliferative disease by administering a compound of the inventionhaving a therapeutic drug moiety derived from a therapeutic drugeffective in treating cell proliferative disease. Representative cellproliferative diseases treatable by the compounds of the inventioninclude hematologic cancers, such as leukemia, lymphoma, and myeloma;and nonhematologic cancers, such as solid tumor carcinomas (e.g.,breast, ovarian, pancreatic, colon, colorectal, non-small cell lung, andbladder), sarcomas, and gliomas.

Therapeutically effective amounts of the compounds will generally rangeup to the maximally tolerated dosage, but the concentrations are notcritical and may vary widely. The precise amounts employed by theattending physician will vary, of course, depending on the compound,route of administration, physical condition of the patient and otherfactors. The daily dosage may be administered as a single dosage or maybe divided into multiple doses for administration.

The amount of the compound actually administered will be atherapeutically effective amount, which term is used herein to denotethe amount needed to produce a substantial beneficial effect. Effectivedoses may be extrapolated from dose-response curves derived from invitro or animal model test systems. The animal model is also typicallyused to determine a desirable dosage range and route of administration.Such information can then be used to determine useful doses and routesfor administration in humans or other mammals. The determination of aneffective dose is well within the capability of those skilled in theart. Thus, the amount actually administered will be dependent upon theindividual to which treatment is to be applied, and will preferably bean optimized amount such that the desired effect is achieved withoutsignificant side-effects.

Therapeutic efficacy and possible toxicity of the compounds of theinvention can be determined by standard pharmaceutical procedures, incell cultures or experimental animals (e.g., ED₅₀, the dosetherapeutically effective in 50% of the population; and LD₅₀, the doselethal to 50% of the population). The dose ratio between therapeutic andtoxic effects is the therapeutic index, and it can be expressed as theratio LD₅₀ to ED₅₀. Modified therapeutic drug compounds that exhibitlarge therapeutic indices are particularly suitable in the practice ofthe methods of the invention. The data obtained from cell culture assaysand animal studies may be used in formulating a range of dosage for usein humans or other mammals. The dosage of such compounds lies preferablywithin a range of circulating concentrations that include the ED₅₀ withlittle or no toxicity. The dosage typically varies within this rangedepending upon the dosage form employed, sensitivity of the patient, andthe route of administration. Thus, optimal amounts will vary with themethod of administration, and will generally be in accordance with theamounts of conventional medicaments administered in the same or asimilar form.

The compounds of the invention can be administered alone, or incombination with one or more additional therapeutic agents. For example,in the treatment of cancer, the compounds can be administered incombination with therapeutic agents including, but not limited to,androgen inhibitors, such as flutamide and luprolide; antiestrogens,such as tomoxifen; antimetabolites and cytotoxic agents, such asdaunorubicin, fluorouracil, floxuridine, interferon alpha, methotrexate,plicamycin, mecaptopurine, thioguanine, adriamycin, carmustine,lomustine, cytarabine, cyclophosphamide, doxorubicin, estramustine,altretamine, hydroxyurea, ifosfamide, procarbazine, mutamycin, busulfan,mitoxantrone, carboplatin, cisplatin, streptozocin, bleomycin,dactinomycin, and idamycin; hormones, such as medroxyprogesterone,estramustine, ethinyl estradiol, estradiol, leuprolide, megestrol,octreotide, diethylstilbestrol, chlorotrianisene, etoposide,podophyllotoxin, and goserelin; nitrogen mustard derivatives, such asmelphalan, chlorambucil, methlorethamine, and thiotepa, steroids, suchas betamethasone; and other antineoplastic agents, such as liveMycobacterium bovis, dicarbazine, asparaginase, leucovorin, mitotane,vincristine, vinblastine, and taxotere. Appropriate amounts in each casewill vary with the particular agent, and will be either readily known tothose skilled in the art or readily determinable by routineexperimentation.

Administration of the compounds of the invention is accomplished by anyeffective route, for example, parenteral, topical, or oral routes.Methods of administration include inhalational, buccal, intramedullary,intravenous, intranasal, intrarectal, intraocular, intraabdominal,intraarterial, intraarticular, intracapsular, intracervical,intracranial, intraductal, intradural, intralesional, intramuscular,intralumbar, intramural, intraocular, intraoperative, intraparietal,intraperitoneal, intrapleural, intrapulmonary, intraspinal,intrathoracic, intratracheal, intratympanic, intrauterine,intravascular, and intraventricular administration, and otherconventional means. The compounds of the invention having anti-tumoractivity can be injected directly into a tumor, into the vicinity of atumor, or into a blood vessel that supplies blood to the tumor.

The emulsion, microemulsion, and micelle formulations of the inventioncan be nebulized using suitable aerosol propellants that are known inthe art for pulmonary delivery of the compounds.

The compounds of the invention may be formulated into a composition thatadditionally comprises suitable pharmaceutically acceptable carriers,including excipients and other compounds that facilitate administrationof the compound to a subject. Further details on techniques forformulation and administration may be found in the latest edition of“Remington's Pharmaceutical Sciences” (Maack Publishing Co., Easton,Pa.).

Compositions for oral administration may be formulated usingpharmaceutically acceptable carriers well known in the art, in dosagessuitable for oral administration. Such carriers enable the compositionscontaining the compounds of the invention to be formulated as tablets,pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions,suitable for ingestion by a subject. Compositions for oral use may beformulated, for example, in combination with a solid excipient,optionally grinding the resulting mixture, and processing the mixture ofgranules, after adding suitable additional compounds, if desired, toobtain tablets or dragee cores. Suitable excipients include carbohydrateor protein fillers. These include, but are not limited to, sugars,including lactose, sucrose, mannitol, or sorbitol, starch from corn,wheat, rice, potato, or other plants; cellulose such as methylcellulose, hydroxypropylmethyl-cellulose, or sodiumcarboxymethylcellulose; and gums including arabic and tragacanth; aswell as proteins, such as gelatin and collagen. If desired,disintegrating or solubilizing agents may be added, such as thecrosslinked polyvinyl pyrrolidone, agar, alginic acid, or a saltthereof, such as sodium alginate.

Dragee cores are provided with suitable coatings such as concentratedsugar solutions, which may also contain gum arabic, talc,polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and/or titaniumdioxide, lacquer solutions, and suitable organic solvents or solventmixtures. Dyestuffs or pigments may be added to the tablets or drageecoatings for product identification or to characterize the quantity ofactive compound (i.e., dosage).

Compounds for oral administration may be formulated, for example, aspush-fit capsules made of gelatin, as well as soft, sealed capsules madeof gelatin and a coating such as glycerol or sorbitol. Push-fit capsulesmay contain the compounds mixed with filler or binders such as lactoseor starches, lubricants such as talc or magnesium stearate, and,optionally, stabilizers. In soft capsules, the covalent conjugates maybe dissolved or suspended in suitable liquids, such as fatty oils,liquid paraffin, or liquid polyethylene glycol with or withoutstabilizers.

For topical or nasal administration, penetrants appropriate to theparticular barrier to be permeated are typically used in theformulation. Examples of these are 2-pyrrolidone,N-methyl-2-pyrrolidone, dimethylacetamide, dimethyl-formamide, propyleneglycol, methyl or isopropyl alcohol, dimethyl sulfoxide, and azone.Additional agents may further be included to make the formulationcosmetically acceptable. Examples of these are fats, waxes, oils, dyes,fragrances, preservatives, stabilizers, and surface-active agents.Keratolytic agents such as those known in the art may also be included.Examples are salicylic acid and sulfur. For topical administration, thecomposition may be in the form of a transdermal ointment or patch forsystemic delivery of the compound and may be prepared in a conventionalmanner (see, e.g., Barry, Dermatological Formulations (Drugs and thePharmaceutical Sciences—Dekker); Harry's Cosmeticology (Leonard HillBooks).

For rectal administration, the compositions may be administered in theform of suppositories or retention enemas. Such compositions may beprepared by mixing the compounds with a suitable non-irritatingexcipient that is solid at ordinary temperatures but liquid at therectal temperature and will therefore melt in the rectum to release thedrug. Suitable excipients include, but are not limited to, cocoa butterand polyethylene glycols.

The amounts of each of these various types of additives will be readilyapparent to those skilled in the art, optimal amounts being the same asin other, known formulations designed for the same type ofadministration.

Compositions containing the compounds of the invention may bemanufactured in a manner similar to that known in the art (e.g., bymeans of conventional mixing, dissolving, granulating, dragee-making,levigating, emulsifying, encapsulating, entrapping or lyophilizingprocesses). The compositions may also be modified to provide appropriaterelease characteristics, sustained release, or targeted release, byconventional means (e.g., coating). As noted above, in one embodiment,the compounds are formulated as an emulsion.

Compositions containing the compounds may be provided as a salt and canbe formed with many acids, including but not limited to hydrochloric,sulfuric, acetic, lactic, tartaric, malic, and succinic. Salts tend tobe more soluble in aqueous or other protonic solvents than are thecorresponding free base forms.

After compositions formulated to contain a compound and an acceptablecarrier have been prepared, they can be placed in an appropriatecontainer and labeled for use. Thus, in another aspect, the inventionprovides kits.

Tocopherol-modified therapeutic drug compounds of the invention aresuitable for administration as oil-in-water emulsions and micelleformulations. The compounds provide for high drug loading to enablesmall volumes for administration.

Emulsions containing tocopherol-modified camptothecin compounds of theinvention provide for enhanced stability of the compound's lactonecompared to conventional methods of camptothecin administration. Longplasma half-life is achieved for the tocopherol-modified camptothecincompounds resulting in prolonged exposure of a tumor to the compounds.Tocopherol-modified compounds achieve high permeation through lipoidalmembranes of tumor cells. Greater anti-tumor response without anincrease in toxicity may be provided by the tocopherol-modifiedcamptothecin compounds of the invention as compared to unmodifiedcamptothecin and currently available camptothecin analogs.

Although the compounds of the invention having formula (2) with m=1,formula (3) with n=1, or formula (8) do not include compoundsspecifically excluded as described above, it will be appreciated thatthe compositions, emulsion formulations, microemulsion formulations, andmicelle formulations include the compounds of the invention havingformulae (1)-(8) without such limitation. Methods for administering thecompositions, emulsion formulations, microemulsion formulations, andmicelle formulations, and methods for treating a condition treatable byadministering the compositions, emulsion formulations, microemulsionformulations, and micelle formulations are likewise not limited withregard to the compounds of the invention.

The following examples are provided to illustrate, not limit, theinvention.

EXAMPLES Example 1 The Preparation of a RepresentativeTocopherol-Modified Camptothecin Compound: Tocopherol SuccinateCamptothecin

A 500 ml flask was charged with 10.6 grams of d-α-tocopherol succinicacid, 6.97 grams of camptothecin, 6.13 grams of2-chloro-1-methylpyridinium iodide (CMPI), 5.86 grams of4-(dimethylamino)pyridine (DMAP), and 200 ml of dryN,N-dimethylacetamide. The mixture was stirred at room temperature for24 hours, and then heated at 50° C. for 4 hours. The mixture was cooledto room temperature and then was filtered to remove precipitate and thefiltrate was collected. To the filtrate were added 250 ml of chloroformand 150 ml of deionized-water to extract the product into thechloroform, and the water fraction was removed using a separationfunnel. The chloroform fraction was washed with deionized-water (3×150ml) in a separation funnel, collected, and dried over anhydrous MgSO₄overnight. The MgSO₄ was removed by filtration, and the chloroform wasremoved with a rotary evaporator under reduced pressure to yield adark-yellow solid. The product was purified by column chromatography onsilica gel. (Yield: 9.50 grams, 55.2%).

¹H NMR (300 MHz, CDCl₃): δ 8.318 (s, 1H), 8.163-8.135 (d, J=8.4 Hz, 1H),7.927-7.901(d, J=7.8 Hz, 1H), 7.842-7.787 (m, 1H), 7.682-7.632 (m, 1H),7.263-7.242 (d, J=6.3 Hz, 1H), 5.702-5.410 (ABq, J₁=17.4, J₂=70 Hz, 2H),5.190 (s, 2H), 3.014-2.938 (m, 4H), 2.368-0.809 (m, 54H).

Elemental anal. Calcd. for C₅₃H₆₈N₂O₈: C, 73.92; H, 7.96; N, 3.25.Found: C, 73.61; H, 7.90; N, 3.17.

Example 2 The Preparation of a Representative Tocopherol-ModifiedCamptothecin Compound: Tocopherol Succinate 10-Hydroxycamptothecin

Method 1. A 100 ml flask was charged with 1.06 grams of d-α-tocopherolsuccinic acid, 0.476 grams of thionyl chloride, and 50 ml of toluene.The mixture was stirred at room temperature overnight. The solvent wasremoved with a rotary evaporator at 50° C., and the residue wascollected. To the residue was added 0.728 grams of10-hydroxycamptothecin and 40 ml of dried tetrahydrofuran with stirring.Then, 0.404 grams of triethylamine in 10 ml of tetrahydrofuran was addeddropwise to the reaction mixture. The mixture was stirred at roomtemperature overnight. The mixture was filtered and white powder waswashed with ethyl acetate (3×10 ml). The filtrate was collected. Thesolvent was removed with a rotary evaporator. The residue was collected,and purified by column chromatography on silica gel with a mobile phaseof acetone and chloroform (1:4, v/v). (Yield: 0.85 grams, 48.4%).

MS (Positive ESI): m/z 877 (M)⁺.

Anal. Calcd. for C₅₃H₆₈N₂O₉: C, 72.58; H, 7.81; N, 3.19. Found: C,72.52; H, 7.84; N, 3.21.

Alternatively, tocopherol succinate 10-hydroxycamptothecin can beprepared as described below.

Method 2. A 100 ml flask was charged with 2.65 grams of d-α-tocopherolsuccinate, 0.89 grams of thionyl chloride, and 20 ml of toluene. Themixture was stirred at room temperature for 24 hours. The toluene andany excess thionyl chloride were removed with vacuum distillation at 50°C. The remaining residue was dissolved in 15 ml of chloromethane toprovide Solution A. To a 100 ml flask, 0.9 grams of10-hydroxycamptothecin, 0.5 ml of triethylamine, and 25 ml of freshlydried N,N-dimethylacetamide was added with stirring. Then 15 ml ofSolution A was slowly added into the mixture through a dropping funnelover 5 minutes. The reaction mixture was stirred at room temperature for24 hours. The mixture was concentrated by vacuum distillation. 150 ml ofethyl acetate was added to the residue. The mixture was washed withsaturated aqueous NaCl solution, (3×100 ml). The mixture was dried overanhydrous MgSO₄. The MgSO₄ was removed by filtration and the ethylacetate was then removed by vacuum distillation. The crude product waspurified by column chromatography on silica gel. (Yield: 1.14 grams,52.5%).

Example 3 The Preparation of a Representative Tocopherol-ModifiedCamptothecin Compound: Tocopherol Succinate7-Ethyl-10-hydroxycamptothecin

Method 1. A 500 ml flask was charged with 22.5 grams of d-α-tocopherolsuccinate, 7.6 grams of thionyl chloride, and 200 ml of toluene. Themixture was stirred at room temperature for 24 hours. The toluene andthe excess thionyl chloride were removed by vacuum distillation. Theremaining residue was dissolved in 100 ml of chloromethane to provideSolution A. Solution A was used immediately, and was not exposed to air.To a 500 ml flask, 7.8 grams of 7-ethyl-10-hydroxycamptothecin, 7 ml oftriethylamine, and 250 ml of freshly dried N,N-dimethylacetamide wasadded with stirring. The 100 ml of Solution A was slowly added into themixture through a dropping funnel over 30 minutes. The reaction mixturewas stirred at room temperature for 24 hours. The solvent wasconcentrated by vacuum distillation. 500 ml of ethyl acetate was addedto the residue. The mixture was washed with saturated aqueous NaClsolution, (3×200 ml). The mixture was dried over anhydrous MgSO₄. TheMgSO₄ was removed by filtration and the ethyl acetate was then removedby vacuum distillation. The crude product was purified byrecrystallization with acetone. (Yield: 15.18 grams, 83.9%).

M.P. 171°-173° C.

¹H NMR (300 MHz, CDCl₃): δ 8.236-8.206 (d, J=9 Hz, 1H), 7.809-7.801 (d,J=2.4 Hz, 1H), 7.648 (s, 1H), 7.572-7.533 (dd, J₁=2.7 Hz, J₂=9.3 Hz,1H), 5.781-5.280 (ABq, J₁=16.2 Hz, J₂=134.0 Hz, 2H), 5.253 (s, 2H),3.863 (s, 1H), 3.136-3.113 (m, 6H), 2.588 (t, 2H), 2.091 (s, 3H), 2.037(s, 3H), 1.994 (s, 3H), 1.970-1.852 (m, 2H), 1.821-1.725 (m, 2H),1.654-0.833 (m, 42H).

MS (Positive ESI): m/z 905 (M)⁺, 928 (M+Na)⁺.

Anal. Calcd. for C₅₅H₇₂N₂O₉: C, 72.98; H, 8.02; N, 3.09. Found: C,72.87; H, 8.01; N, 2.88.

Alternatively, the tocopherol succinate 7-ethyl-10-hydroxycamptothecincan be prepared as described below.

Method 2. A 500 ml flask was charged with 8.48 grams of d-α-tocopherolsuccinate, 3.81 grams of thionyl chloride, and 250 ml of toluene. Themixture was stirred at room temperature overnight. The toluene, andexcess thionyl chloride were removed with a rotary evaporator at 50° C.,and the residue was collected. To the residue was added 6.27 grams of7-ethyl-10-hydroxycamptothecin and 250 ml of sodium-driedtetrahydrofuran with stirring. Then, 3.23 grams of triethylamine in 50ml of tetrahydrofuran was added dropwise to the mixture. The mixture wasstirred at room temperature overnight. The mixture was filtered and thewhite powder was washed with ethyl acetate (3×50 ml). The filtrate wascollected. The solvent was removed with a rotary evaporator. The crudeproduct was purified by recrystallization in acetone. (Yield: 8.28grams, 57.2%).

Example 4 The Preparation of a Representative Tocopherol-ModifiedCamptothecin Compound: 10,20-Di(tocopherol succinate)7-Ethyl-10-hydroxycamptothecin

A 100 ml flask was charged with 0.905 grams of tocopherol succinate7-ethyl-10-hydroxycamptothecin, 0.53 grams of d-α-tocopherol succinicacid, 0.255 grams of 2-chloro-1-methylpyridinium iodide, 0.244 grams of4-(dimethylamino)pyridine and 50 ml of dioxane. The mixture was stirredat room temperature for 24 hours. Thin layer chromatography showed thatthe reaction was complete. The mixture was filtered to remove the solidphase, and the filtrate was collected. The solvent was removed by vacuumdistillation. The crude product was purified by column chromatography onsilica gel with 30% ethyl acetate in cyclohexane. (Yield: 0.64 grams,44.82%).

¹H NMR (300 MHz, CDCl₃): δ 8.168-8.138 (d, J=9.0 Hz, 1H), 7.813-7.805(d, J=2.4 Hz, 1H), 7.754-7.536 (dd, J₁=2.1 Hz, J₂=11.4 Hz, 1H), 7.197(s, 1H), 5.703-5.409 (ABq, J₁=17.4 Hz, J₂=71.0 Hz, 2H), 5.243-5.088 (m,2H), 3.113-2.857 (m, 10H), 2.606-2.564 (t, J=6 Hz, 2H), 2.383-2.184 (m,2H), 2.090-1.723 (m, 22H), 1.588-0.785 (m, 80H).

MS (Positive ESI): m/z 1418 (M+H)⁺.

Elemental anal. Calcd. for C₈₈H₁₂₄N₂O₁₃: C, 74.54; H, 8.81; N, 1.98.Found: C, 74.31; H, 8.96; N, 1.75.

IRν_(max) ^(KBr)cm⁻¹: 2925, 2867, 1751, 1665, 1615, 1657, 1510, 1458,1413, 1376, 1330, 1218, 1128, 1075, 1060, 1035, 992, 943, 923, 829, 812,758, 724, 668.

Example 5 The Preparation of a Representative Tocopherol-ModifiedCamptothecin Compound: Tocopherol-Camptothecin Conjugate with aHexa(ethylene glycol) Linker

Preparation of hexa(ethylene glycol) tocopherol succinate. In a 250 mlflask, 2.65 grams of d-α-tocopherol succinic acid and 2.82 grams ofhexa(ethylene glycol) was dissolved in 100 ml of toluene with stirring.The toluene was removed with a rotary evaporator (drying by azeotropicdistillation). To the mixture was added 100 ml of chloroform, 1.08 gramsof N,N-dicyclohexylcarbodiimide, and 100 mg of4-(dimethylamino)pyridine. The mixture was stirred overnight. Thin layerchromatography with 40% acetone in hexane showed that the reaction wascomplete. The mixture was washed three times with deionized-water (3×100ml), and the chloroform fraction was collected, and dried over anhydrousMgSO₄ for two hours. After filtration, chloroform was removed by arotary evaporator. The crude product was purified by columnchromatography on silica gel using, successively, the solvents 30% ethylacetate in hexane, 50% ethyl acetate in hexane, and 30% acetone inhexane. (Yield: 0.53 grams, 13.33%).

Preparation of tocopherol-succinyl-hexa(ethylene glycol) succinic acid.A 100 ml flask was charged with 1.42 grams of hexa(ethylene glycol)tocopherol succinate prepared above, 0.2 grams of succinic acidanhydride, 2 drops of tin (II) ethylhexanoate, and 25 ml of xylene. Themixture was refluxed for 4 hours. After the reaction was complete, thesolvent was removed by a rotary evaporator. The crude product waspurified by column chromatography on silica gel. (Yield: 0.864 grams,54%).

Preparation of tocopherol succinate camptothecin with a hexa(ethyleneglycol) linker. A 100 ml flask was charged with 0.822 grams oftocopherol-succinyl-hexa(ethylene glycol) succinic acid prepared above,0.3 grams of camptothecin, 0.47 grams of 2-chloro-1-methylpyridiniumiodide, 0.45 grams of 4-(dimethylamino)pyridine, and 40 ml of driedN,N-dimethylacetamide. The reaction mixture was stirred at roomtemperature overnight. After the reaction was complete, the solvent wasremoved by vacuum distillation, and the residue was collected. To theresidue was added 100 ml of ethyl acetate. After stirring for 30minutes, the mixture was filtered to remove precipitate, and thefiltrate was collected and concentrated. The crude product was purifiedby column chromatography on silica gel. (Yield: 0.342 grams, 30.4%).

¹H NMR (300 MHz, CDCl₃): δ 8.388 (s, 1H), 8.240-8.213 (d, J=8.1 Hz, 1H),7.951-7.923 (d, J=8.4 Hz, 1H), 7.861-7.805 (dt, J₁=1.5 Hz, J₂=8.4 Hz,1H), 7.694-7.640 (dt, J₁=1.2 Hz, J₂=8.1 Hz, 1H), 7.269 (s, 1H),5.708-5.365 (ABq, J₁=17.1 Hz, J₂=85.8 Hz, 2H), 5.283 (s, 2H),4.273-4.169 (m, 4H), 3.705-3.673 (t, 2H), 3.631-3.550 (m, 18H),2.926-2.654 (m, 8H), 2.597-2.552 (t, J=6.9 Hz, 2H), 2.381-2.2.113 (m,2H), 2.074 (s, 3H), 2.004 (s, 3H), 1.964 (s, 3H), 1.827-1.683 (m, 2H),1.655 (s, 3H), 1.544-0.964 (m, 24H), 0.875-0.830 (m, 12H).

MS (Positive ESI): m/z 1225 (M)⁺.

Elemental anal. Calcd. for C₈₈H₁₂₄N₂O₁₃: C, 67.62; H, 7.90; N, 2.29.Found: C, 67.08; H, 8.04; N, 2.07.

IRν_(max) ^(KBr)cm⁻¹: 2925, 2867, 1735, 1667, 1618, 1563, 1500, 1457,1405, 1366, 1349, 1232, 1204 1141, 1107, 1060, 994, 945, 859, 813, 787,761, 723, 707.

Example 6 The Preparation of a Representative Tocopherol-ModifiedPaclitaxel Compound: Tocopherol Succinate Paclitaxel

A 250 ml flask was charged with 5.83 grams of tocopherol succinic acid,2.38 grams of thionyl chloride, and 50 ml of toluene. The mixture wasstirred at room temperature overnight. The solvent was removed with arotary evaporator at 50° C., and the residue was collected. To theresidue were added 8.54 grams of paclitaxel and 100 ml of driedtetrahydrofuran with stirring. Then, 1.52 grams of triethylamine in 50ml of tetrahydrofuran was added dropwise to the reaction mixture. Themixture was stirred at room temperature overnight. The mixture wasfiltered and the white powder was washed with ethyl acetate (3×10 ml).The filtrate was collected. The solvent was removed with a rotaryevaporator. The residue was collected, and purified by recrystallizationin acetone and hexane. (Yield: 11.56 grams, 84.6%).

Anal. Calcd. for C₈₀H₁₀₃NO₁₈: C, 70.31; H, 7.59; N, 1.02. Found: C,70.02, H, 7.83; N, 0.93.

Example 7 The Preparation of a Representative Tocopherol-ModifiedDocetaxel Compound: Tocopherol Succinate Docetaxel

A 250 ml flask is charged with 9.86 grams of d-α-tocopherol succinicacid, 5.0 grams of docetaxel, 3.83 grams of driedN,N-dicyclohexylcarbodiimide, 500 mg of 4-(dimethylamino)pyridine, and150 ml of chloroform. The mixture is stirred at room temperatureovernight. The mixture is filtered to remove precipitate and thefiltrate is collected. The solvent is removed with rotary evaporator,and the residue is collected. The crude product is purified by columnchromatography on silica gel.

Example 8 The Preparation of Mono-Tocopherol Phthalate

A 100 mL flask was charged with 8.61 grams of dl-α-tocopherol, 2.96grams of phthalic anhydride, 50 mg of tin (II) 2-ethylhexanoate, and 50ml of dried N,N-dimethylacetamide. The mixture was stirred at about 140°C. for 24 hours. After the mixture was cooled to room temperature, themixture was poured into 150 ml of ethyl acetate. The mixture was washedthree times with saturated aqueous NaCl (3×100 ml), and dried overanhydrous MgSO₄ overnight. The crude product was purified by columnchromatography on silica gel with 30% ethyl acetate in hexane. (Yield:3.6 grams, 31.1%).

¹H NMR (300 MHz, CDCl₃) δ ppm: 10.80 (bs, 1H), 8.119-8.063 (m, 1H),7.883-7.828 (m, 1H), 7.678-7.616 (m, 2H), 2.627-2.582 (t, 2H), 2.123 (s,3H), 2.112 (s, 3H), 2.081 (s, 3H), 1.868-1.702 (m, 2H), 1.616-1.020 (m,24H), 0.874-0.834 (m, 12H).

Anal. Calcd. for C₃₇H₅₄O₅: C, 76.78; H, 9.40. Found: C, 76.57; H, 9.29.

IRν_(max) ^(KBr)cm⁻¹: 3073, 2919, 2858, 1737, 1701, 1578, 1455, 1409,1373, 1276, 1230, 1107, 1071, 913, 738.

Example 9 The Preparation of Mono-Tocopherol Terephthalate

A 100 mL flask was charged with 4.30 grams of dl-α-tocopherol, 3.32grams of terephthalic acid, 2.55 grams of 2-chloro-1-methylpyridiniumiodide, 0.244 grams of 4-(dimethylamino)pyridine, and 50 ml of dryN,N-dimethylacetamide. The mixture was stirred at 50° C. for 4 hours.Thin layer chromatography showed that the reaction was complete. Afterthe mixture was cooled to room temperature, the mixture was poured into150 ml of ethyl acetate. The mixture was washed three times withsaturated aqueous NaCl (3×100 mL), and dried over anhydrous MgSO₄overnight. The crude product was purified by column chromatography onsilica gel with 30% ethyl ether in hexane. (Yield: 1.60 grams, 27.6%)

¹H NMR (300 MHz, CDCl₃) δ ppm: 11.80 (bs, 1H), 8.374-8.259 (q, J₁=8.4Hz, J₂=26.1 Hz, 4H), 2.650-2.607 (t, 2H), 2.130 (s, 3H), 2.066 (s, 3H),2.024 (s, 3H), 1.895-1.783 (m, 2H), 1.532-1.083 (m, 24H), 0.878-0.839(m, 12H).

Anal. Calcd. for C₃₇H₅₄O₅: C, 76.78; H, 9.40. Found: C, 76.64; H, 9.39.

IRν_(max) ^(KBr)cm⁻¹: 3062, 2924, 2858, 1737, 1696, 1573, 1460, 1424,1373, 1276, 1240, 1097, 928, 774, 723.

Example 10 The Preparation of a Representative Tocopherol-ModifiedCamptothecin Compound: Tocopherol Terephthalate Camptothecin

A 100 mL flask was charged with 1.16 grams of mono-tocopherolterephthalate prepared above, 0.70 grams of camptothecin, 0.511 grams of2-chloro-1-methylpyridinium iodide, and 0.489 grams of4-(dimethylamino)pyridine. The mixture was stirred at 50° C. overnight.Thin layer chromatography showed the reaction was complete. After themixture cooled to room temperature, the reaction mixture was poured into150 ml of ethyl acetate. The mixture was filtered and the filtrate wascollected. The filtrate was washed with saturated aqueous NaCl (3×100ml), and dried over anhydrous MgSO₄ overnight. The crude product waspurified by column chromatography on silica gel. (Yield: 0.560 grams,30.8%)

¹H NMR (300 MHz, CDCl₃) δ ppm: 8.387 (s, 1H), 8.370-8.242 (q, J₁=8.4 Hz,J₂=30.3 Hz, 4H), 8.167-8.139 (d, J=8.4 Hz, 1H), 7.937-7.910 (d, J=8.1Hz, 1H), 7.823-7.774 (t, 1H), 7.672-7.625 (t, 1H), 7.260 (s, 1H),5.823-5.462 (ABq, J₁=17.4 Hz, J₂=90.9 Hz, 2H), 5.302 (s, 2H), 2.461 (t,2H), 2.559-2.312 (m, 2H), 2.123 (s, 3H), 2.056 (s, 3H), 2.015 (s, 3H),1.844-1.801 (m, 2H), 1.629-1.085 (m, 27H), 0.938-0.789 (m, 12H).

Anal. Calcd. for C₅₇H₆₈N₂O₈: C, 75.30; H, 7.54; N, 3.08. Found: C,74.91; H, 7.56; N, 3.02.

IRν_(max) ^(KBr)cm⁻¹: 3057, 2924, 2858, 1757, 1737, 1675, 1614, 1558,1450, 1399, 1266, 1235, 1163, 1102, 1020,723.

Example 11 The Preparation of a Representative Tocopherol-ModifiedCamptothecin Compound: Tocopherol Cyclohexane-1,2-dicarboxylate7-Ethyl-10-hydroxycamptothecin

Preparation of tocopherol cyclohexane-1,2-dicarboxylic acid. A mixtureof 1.54 grams of 1,2-cyclohexanedicarbolic acid anhydride, 8.6 grams ofd-α-tocopherol, 1.34 grams of aluminum trichloride, and 100 ml ofcyclohexane in a 250 ml flask was heated under reflux for about 30minutes. After the mixture cooled to room temperature, it was filtered.The filtrate was washed with a dilute aqueous hydrochloric acid solutionand then dried over anhydrous MgSO₄. The mixture was concentrated, andcrude product was purified by column chromatography on silica gel.(Yield: 3.325 grams, 56.9%).

Preparation of tocopherol cyclohexane-1,2-dicarboxylate7-ethyl-10-hydroxycamptothecin. A mixture of 1.08 grams of tocopherolcyclohexane-1,2-dicarboxylic acid prepared above, 0.44 grams of thionylchloride, and 20 ml of toluene was stirred under nitrogen overnight. Thetoluene and excess thionyl chloride were removed by vacuum distillation,and the residue was dissolved in 10 ml of dichloromethane to provideSolution A. In a 100 ml flask, 0.350 grams of SN38 was dissolved in 25ml of dried N,N-dimethylacetamide to provide Solution B. Solution A and0.186 g of triethylamine were added to Solution B. The mixture wasstirred overnight at room temperature. The crude product was purified bycolumn chromatography on silica gel. (Yield: 0.59 grams, 68.9%).

Example 12 The Preparation of a Representative Tocopherol-ModifiedDoxorubicin Compound: Tocopherol Succinate Doxorubicin

A 100 ml flask is charged with equivalent moles (1 mmole) of tocopherolsuccinic acid, doxorubicin, and N,N-dicyclohexylcarbodiimide, and 50 mlof dry N,N-dimethylacetamide. The mixture is stirred at room temperatureuntil completion of the reaction. The mixture is filtered to removewhite precipitate and the filtrate is collected. The solvent is removedwith a rotary evaporator, and the residue is collected. The product ispurified by either recrystallization or column chromatography on silicagel.

Example 13 The Preparation of a Representative Tocopherol-ModifiedHydroxyzine Compound: Tocopherol Succinate Hydroxyzine

A 100 ml flask is charged with equivalent moles (1 mmole) of tocopherolsuccinic acid, and thionyl chloride, and 50 ml of toluene. The mixtureis stirred at room temperature overnight. The solvent is removed with arotary evaporator at 50° C., and the residue is collected. To theresidue are added 1 mmole of hydroxyzine and 40 ml of chloroform withstirring. Then, 1 mmole of triethylamine in 10 ml of chloroform is addeddropwise to the reaction mixture at 0-5° C. The mixture is then stirredat room temperature overnight. The mixture is washed with saturatedNaHCO₃ aqueous solution (3×50 ml). The organic phase is collected, anddried with anhydrous MgSO₄. The solvent is removed with a rotaryevaporator after removal of MgSO₄. The residue is collected, and thecrude product is purified by either recrystallization or silica columnchromatography.

Example 14 Representative Tocopherol-Modified Therapeutic Drug CompoundSolubility

In this example, the solubility of representative tocopherol-modifiedtherapeutic drug compounds of the invention, tocopherol succinatecamptothecin and tocopherol succinate 7-ethyl-10-hydroxycamptothecin,was compared to the solubility of camptothecin in a variety of solvents.

The solubility of camptothecin, tocopherol succinate camptothecin, andtocopherol succinate 7-ethyl-10-hydroxycamptothecin was determined inseveral solvents. Compounds were dissolved in each solvent underconstant stirring and temperature to saturation. The resulting solutionswere centrifuged and the supernatant was analyzed by high performanceliquid chromatography (HPLC).

The comparative solubility (mg/g) of camptothecin, tocopherol succinatecamptothecin, and tocopherol succinate 7-ethyl-10-hydroxycamptothecin invarious solvents is shown in Table 1. TABLE 1 Solubility Comparison ofCamptothecin and Tocopherol Succinate Camptothecins. VESA- CamptothecinSN38¹ VESA-CPT² Temperature Solvent (mg/g) (mg/g) (mg/g) (° C.) PEG-400— 0.017 17.5 Room Temp. NF TPGS — 27.6 >133.3 65 Vitamin E 1.96398.2 >288.3 65 USP/NF Soybean Oil 0.00 3.3 45.2 Room Temp. USP Captex300 — 4.4 96.7 Room Temp. EP Tween 80 — 2.7 48.3 65 → Room NF Temp.Ethanol — 4.2 57.1 Room Temp. Denatured Methanol — 3.8 14.4 Room Temp.Acetonitrile 0.09 3.1 49.6 Room Temp. Chloroform 0.71 >97.0 >372.5 RoomTemp. DMSO 50 30.5 >255.5 Room Temp. Methylene 0.9 >99.7 >336.5 RoomTemp. dichloride Propylene — 3.2 0.4633 Room Temp. Glycol, USP Glycerin— 2.8 2.541 Room Temp. USP/EP/BP/JP¹VESA-SN38: tocopherol succinate 7-ethyl-10-hydroxycamptothecin²VESA-CPT: tocopherol succinate camptothecin

The results in Table 1 illustrate that tocopherol succinate camptothecinand tocopherol succinate 7-ethyl-10-hydroxycamptothecin both havesubstantial solubility in oils, and have particularly high solubility invitamin E (α-tocopherol).

Example 15 Representative Tocopherol-Modified Therapeutic DrugCompound-Containing Emulsions

In this example, representative emulsions containing tocopherol-modifiedtherapeutic drug compounds of the invention are described.

A. Tocopherol Succinate 7-Ethyl-10-Hydroxycamptothecin Emulsion

Tocopherol succinate 7-ethyl-10-hydroxycamptothecin, prepared asdescribed in Example 3, was dissolved in vitamin E and then emulsifiedwith the use of a microfluidizer (M110Y Microfluidics) in the presenceof d-α-tocopherol polyethylene glycol 1000 succinate (TPGS), Poloxamer407, and saline to produce an emulsion having the following composition(% by weight): Tocopherol succinate-7-ethyl-10-hydroxycamptothecin 0.69%Vitamin E 7.31% TPGS   5% Poloxamer 407   1% Saline   86%

The emulsion was filtered through a 0.2 μm filter and vialed in sterileglass vials. Mean particle size was approximately 50 nM as determined bysubmicron particle sizer (Nicomp Model 370), with 99% of the particlesless than 80 nm. No evidence of precipitation or loss of concentrationas measured by HPLC was observed for at least 3 months when stored at 4°C.

B. Tocopherol Succinate 7-Ethyl-10-hydroxycamptothecin Emulsion

Tocopherol succinate 7-ethyl-10-hydroxycamptothecin, prepared asdescribed in Example 3, was dissolved in vitamin E and then emulsifiedwith the use of a microfluidizer (M110Y Microfluidics) in the presenceof TPGS and saline to produce an emulsion having the followingcomposition (% by weight): Tocopherolsuccinate-7-ethyl-10-hydroxycamptothecin 0.69% Vitamin E 7.31% TPGS   5%Saline   87%

This formulation resulted in a more yellow and thicker emulsion than theemulsion prepared as described above that included Poloxamer 407. Theemulsion was filtered through a 0.2 μm filter and vialed in sterileglass vials. Mean particle size was approximately 75 nm as determined bysubmicron particle sizer (Nicomp Model 370), with 99% of the particlesless than 170 nm. No evidence of precipitation or loss of concentrationas measured by HPLC was observed for at least 3 months when stored at 4°C.

C. Tocopherol Succinate Camptothecin Emulsion

Tocopherol succinate camptothecin, prepared as described in Example 1,was dissolved in vitamin E and then emulsified with the use of amicrofluidizer (M110Y Microfluidics) in the presence of TPGS, Poloxamer407, and saline to produce an emulsion having the following composition(% by weight): Tocopherol succinate camptothecin 0.74% Vitamin E 7.26%TPGS   5% Poloxamer 407   1% Saline   86%

The emulsion was filtered through a 0.2 μm filter and vialed in sterileglass vials. Mean particle size was approximately 40 nm as determined bysubmicron particle sizer (Nicomp Model 370), with 99% of the particlesless than 75 nm. No evidence of precipitation or loss of concentrationas measured by HPLC was observed for at least 3 months when stored at 4°C.

D. Tocopherol Succinate Camptothecin Emulsion

Tocopherol succinate camptothecin, prepared as described in Example 1,was dissolved in vitamin E and then emulsified with the use of amicrofluidizer (M110Y Microfluidics) in the presence of TPGS, Poloxamer407, and saline to produce an emulsion having the following composition(% by weight): Tocopherol succinate camptothecin 1.48% Vitamin E 6.52%TPGS   5% Poloxamer 407   1% Saline   86%

The emulsion was filtered through a 0.2 μm filter and vialed in sterileglass vials. Mean particle size was approximately 30 nm as determined bysubmicron particle sizer (Nicomp Model 370), with 99% of the particlesless than 100 nm. No evidence of precipitation or loss of concentrationas measured by HPLC was observed for at least 3 months when stored at 4°C.

E. Tocopherol Succinate 7-Ethyl-10-hydroxycamptothecin Emulsion

Tocopherol succinate 7-ethyl-10-hydroxycamptothecin, prepared asdescribed in Example 3, was dissolved in vitamin E and then emulsifiedwith the use of a microfluidizer (M110Y Microfluidics) in the presenceof TPGS and citric acid buffered saline to produce an emulsion havingthe following composition (% by weight): Tocopherolsuccinate-7-ethyl-10-hydroxycamptothecin 0.69% Vitamin E 7.31% TPGS   5%Citric acid buffered saline, pH 3.0   87%

The emulsion was filtered through a 0.2 μm filter and vialed in sterileglass vials. Mean particle size was approximately 60 nm as determined bysubmicron particle sizer (Nicomp Model 370), with 99% of the particlesless than 150 nm. No evidence of precipitation or loss of concentrationas measured by HPLC was observed for at least 3 months when stored at 4°C. and 25° C.

F. Tocopherol Succinate 7-Ethyl-10-hydroxycamptothecin Emulsion

Tocopherol succinate 7-ethyl-10-hydroxycamptothecin, prepared asdescribed in Example 3, was dissolved in vitamin E and then emulsifiedwith the use of a microfluidizer (M110Y Microfluidics) in the presenceof TPGS and succinate buffered saline to produce emulsions having thefollowing composition (% by weight): Formulation 1 Tocopherolsuccinate-7-ethyl-10-hydroxycamptothecin 0.69% Vitamin E 7.31% TPGS   5%Succinate buffered saline, pH 4.0   87%

The emulsion was filtered through a 0.2 μm filter and vialed in sterileglass vials. Mean particle size was approximately 70 nm as determined bysubmicron particle sizer (Nicomp Model 370), with 99% of the particlesless than 170 nm. No evidence of precipitation or loss of concentrationas measured by HPLC was observed for at least 3 months when stored at 4°C. and 25° C. Formulation 2 Tocopherolsuccinate-7-ethyl-10-hydroxycamptothecin 1% Vitamin E 7% TPGS 5%Succinate buffered saline, pH 4.0 87% 

The emulsion was filtered through a 0.2 μm filter and vialed in sterileglass vials. Mean particle size was approximately 70 nm as determined bysubmicron particle sizer (Nicomp Model 370), with 99% of the particlesless than 170 nm. No evidence of precipitation or loss of concentrationas measured by HPLC was observed for at least 1 month when stored at 4°C., 25° C., and 40° C. Formulation 3 Tocopherolsuccinate-7-ethyl-10-hydroxycamptothecin 1% Vitamin E 6% TPGS 4%Succinate buffered saline, pH 4.0 89% 

The emulsion was filtered through a 0.2 μm filter and vialed in sterileglass vials. Mean particle size was approximately 95 nm as determined bysubmicron particle sizer (Nicomp Model 370), with 99% of the particlesless than 220 nm. No evidence of precipitation or loss of concentrationas measured by HPLC was observed for at least 1 month when stored at 4°C., 25° C., and 40° C.

G. Tocopherol Succinate 7-Ethyl-10-hydroxycamptothecin (VESA-SN38)Micelle Formulation

Tocopherol succinate 7-ethyl-10-hydroxycamptothecin was dissolved in amixture containing TPGS, PEG(300), and ethanol at about 50° C.-about 60°C. with stirring for about 1 hour to form a transparent solution. Tothis solution was added either deionized-water (DI-water), Poloxamer 407and DI-water, Poloxamer 188 and DI-water, or 0.9% NaCl aqueous solutionto form Formulations 1-5 respectively below. The formulations werestirred for a few minutes to form transparent micelle solutions havingthe following compositions (% by weight): Formulation 1 VESA-SN38 0.2%  TPGS 5% Ethanol 5% PEG(300) 5% DI-water 84.8%  

The formulation solution was filtered through a 0.2 μm filter and vialedin sterile glass vials. No evidence of precipitation or loss ofconcentration as measured by HPLC was observed for at least 11 weekswhen stored at 4° C. Formulation 2 VESA-SN38 0.2% TPGS   5% Poloxamer407 1.7% Ethanol   5% PEG(300)   5% DI-water 83.1% 

The formulation solution was filtered through a 0.2 μm filter and vialedin sterile glass vials. No evidence of precipitation or loss ofconcentration as measured by HPLC was observed for at least 11 weekswhen stored at 4° C. Formulation 3 VESA-SN38 0.2% TPGS   5% PEG(300)  5% Ethanol   5% Poloxamer 188 1.7% DI-water 83.1% 

The formulation solution was filtered through a 0.2 μm filter and vialedin sterile glass vials. No evidence of precipitation or loss ofconcentration as measured by HPLC was observed for at least 11 weekswhen stored at 4° C., and 25° C. Formulation 4 VESA-SN38 0.2% TPGS   2%PEG(300)   2% Ethanol   4% Saline 91.8% 

The formulation solution was filtered through a 0.2 μm filter and vialedin sterile glass vials. No evidence of precipitation or loss ofconcentration as measured by HPLC was observed for at least 1 week whenstored at 4° C., 25° C., or 40° C. Formulation 5 VESA-SN38 0.5% TPGS  5% PEG(300)   5% Ethanol  10% Saline 79.5% 

The formulation solution was filtered through a 0.2 μm filter and vialedin sterile glass vials. No evidence of precipitation or loss ofconcentration as measured by HPLC was observed for at least 3 weeks whenstored at 4° C.

H. Tocopherol Succinate 7-Ethyl-10-hydroxycamptothecin (VESA-SN38)Micelle Formulation

Tocopherol succinate 7-ethyl-10-hydroxycamptothecin was dissolved in amixture containing TPGS, PEG(300), and ethanol at about 50° C.-about 60°C. with stirring for about 1 hour to form a transparent solution. Tothis solution was added succinate buffered saline to form Formulations 1and 2 below. The formulations were stirred for a few minutes to formtransparent micelle solutions having the following compositions (% byweight): Formulation 1 VESA-SN38 0.2% TPGS   2% Ethanol   4% PEG(300)  2% Succinate buffered saline, pH 4.0 91.8% 

The formulation solution was filtered through a 0.2 μm filter and vialedin sterile glass vials. Formulation 2 VESA-SN38 0.5% TPGS   5% Ethanol 10% PEG(300)   5% Succinate buffered saline, pH 4.0 79.5% 

The formulation solution was filtered through a 0.2 μm filter and vialedin sterile glass vials.

Example 16 In vitro Stability of Lactone of RepresentativeTocopherol-Modified Therapeutic Drug Compounds in Presence of HumanAlbumin

In this example, the in vitro stability in the presence of human albuminof the lactone form of representative tocopherol-modified therapeuticdrug compounds of the invention, tocopherol succinate camptothecin andtocopherol succinate 7-ethyl-10-hydroxycamptothecin, was compared to thein vitro stability of the lactone form of camptothecin.

Because the lactone (ring E) is a critical moiety for camptothecinactivity and it is reported not to be stable under physiologicalconditions (pH=7.4), the stability of the lactone for tocopherolsuccinate camptothecin and tocopherol succinate7-ethyl-10-hydroxycamptothecin was determined. The solubilization oftocopherol succinate camptothecin and tocopherol succinate7-ethyl-10-hydroxycamptothecin in the oil-phase is thought to protectthe lactone from hydrolysis and thus provide improved lactone stabilityin physiological conditions. To evaluate lactone stability, a salinebuffered solution (10 mM, pH 7.4) containing 4% human serum albumin wasincubated at 37° C. in the presence of camptothecin (dissolved in DMSO),tocopherol succinate camptothecin emulsion (prepared as described inExample 15C, referred to herein as “SN2300 emulsion”) or tocopherolsuccinate 7-ethyl-10-hydroxycamptothecin emulsion (prepared as describedin Example 15A, referred to herein as “SN2310 emulsion”). Highperformance liquid chromatography with fluorescence detection was usedto analyze the decrease in the concentration of the lactone form overtime.

FIG. 12 illustrates the percent change in concentration of the lactoneform over time for camptothecin, tocopherol succinate camptothecin(SN2300), and tocopherol succinate 7-ethyl-10-hydroxycamptothecin(SN2310), in the presence of human serum albumin. The stability of thelactone of tocopherol succinate camptothecin and tocopherol succinate7-ethyl-10-hydroxycamptothecin is greater than that of camptothecin.This dramatic increase in the stability of the lactone may result inincreased activity compared to the unmodified camptothecin parentcompound.

Example 17 In vitro Cytotoxicity of Representative Tocopherol-ModifiedTherapeutic Drug Compounds

In this example, the in vitro cytotoxicty of representativetocopherol-modified therapeutic drug compounds of the invention,tocopherol succinate camptothecin and tocopherol succinate7-ethyl-10-hydroxycamptothecin, was compared to the in vitrocytotoxicity of camptothecin, 10-hydroxycamptothecin, SN38, irinotecan,and topotecan.

The in vitro cytotoxicity, as measured by GI₅₀ (50% of growthinhibition) values, of tocopherol succinate camptothecin and tocopherolsuccinate 7-ethyl-10-hydroxycamptothecin was investigated and comparedto the National Cancer Institute (NCI) GI₅₀ values for camptothecin,10-hydroxycamptothecin, SN-38, irinotecan, and topotecan in thefollowing cancer cell lines: NCI-H460 (ATCC #HTB-177) (non-small celllung), HCT-15 (ATCC #CCL-225) (colorectal), HT-116 (ATCC #CCL-247)(colorectal), HT-29 (ATCC #HTB-38) (colorectal), MCF-7 (ATCC #HTB-22)(breast), and OVCAR-3 (ATCC #HTB-161) (ovarian).

The study was performed using emulsion formulations of tocopherolsuccinate 7-ethyl-10-hydroxycamptothecin (described in Example 15A) andtocopherol succinate camptothecin (described in Example 15C) diluted inthe corresponding cell media. The cells were in contact with varyingconcentrations of the test article for a period of 48 hours. At the endof 48 hours, staining with ALAMAR BLUE was performed to determine thenumber of viable cells and calculate the degree of cellular growthinhibition as compared to a control group. The percent of inhibitionversus concentration was fit to the Hill equation to determineconcentration that produces 50% of growth inhibition (GI₅₀).

The sensitivity of the tested cell lines to tocopherol succinatecamptothecin (SN2300), tocopherol succinate7-ethyl-10-hydroxycamptothecin (SN2310), camptothecin, irinotecan, andtopotecan is illustrated in Table 2 and FIG. 13. TABLE 2 Comparativedrug concentration that produce 50% cell growth inhibition (GI₅₀).10-HO- CPT CPT SN38 Irinotecan Topotecan VESA- VESA- Cell line (NCI)(NCI) (NCI) (NCI) (NCI) CPT SN38 NCI-H460 16 nM 11 nM 1.4 nM 5.01 μM19.9 nM 43 nM 4 nM (NSCLC) HCT-15 160 nM 356 nM 7.9 nM 31.6 μM 501 nM 20μM 99 nM (COLON) OVCAR-3 160 nM 62 nM 2.9 nM 31.6 μM 251 nM Poor 83 nM(OVARIAN) activity HCT-116 40 nM 27 nM 21 nM 7.9 μM 39.8 nM 449 nM 119nM (COLON) HT29 126 nM 112 nM 1 nM 12.58 μM 125 nM 434 nM 91 nM (COLON)MCF-7 13 nM 10 nM 3.98 μM 15.8 nM 325 nM (BREAST)CPT: Camptothecin;10-HO-CPT: 10-hydroxycamptothecin;SN38: 7-ethyl-10-hydroxycamtothecin;VESA-CPT: tocopherol succinate camptothecin;VESA-SN38: tocopherol succinate 7-ethyl-10-hydroxycamothecin.

The results in Table 2 illustrates that formulations oftocopherol-modified therapeutic drug compounds of the invention provideeffective anti-tumor activity.

FIG. 13 is a plot of the GI₅₀ values (concentration that produces 50%growth inhibition) determined for tocopherol succinate camptothecin andtocopherol succinate 7-ethyl-10-hydroxycamptothecin in four of thetested cell lines. The values reported by the NCI in these same cancercell lines for camptothecin, irinotecan, and topotecan is also includedas comparison. A high GI₅₀ value corresponds to a low drug concentrationto produce 50% inhibition. From the graph, it is clear that thecompounds of the invention, tocopherol succinate camptothecin andtocopherol succinate 7-ethyl- 10-hydroxycamptothecin, show a high levelof cytotoxic activity similar to camptothecin.

Example 18 Pharmacokinetics of Representative Tocopherol-ModifiedTherapeutic Drug Compounds

In this example, the pharmacokinetics of representativetocopherol-modified therapeutic drug compounds of the invention,tocopherol succinate camptothecin and tocopherol succinate7-ethyl-10-hydroxycamptothecin, was compared to camptothecin,irinotecan, and topotecan.

The pharmacokinetic profiles of tocopherol succinate camptothecin andtocopherol succinate 7-ethyl-10-hydroxycamptothecin were investigated inSprague-Dawley rats following a bolus, intravenous administration ofemulsion formulations of the drug compounds (SN2300 emulsion and SN2310emulsion) via the lateral tail vein at a dose of approximately 14 mg ofdrug compound/kg of body weight. Blood samples were collected for up to120 hours post dose via the jugular vein. The concentration of eachcamptothecin derivative in plasma was determined by high performanceliquid chromatography (HPLC) with fluorescence detection. Anoncompartmental analysis was carried out using WinNonlin (v 4.1).

The pharmacokinetic profiles of tocopherol succinate camptothecin(SN2300) and tocopherol succinate 7-ethyl-10-hydroxycamptothecin(SN2310) are illustrated in FIGS. 14A and 14B, respectively. FIGS. 14Aand 14B illustrate concentration-time values after an intravenousinjection of 13.8 mg of drug compound/kg of body weight for tocopherolsuccinate camptothecin (SN2300 emulsion) and tocopherol succinate7-ethyl-10-hydroxycamptothecin (SN2310 emulsion), respectively.Referring to FIGS. 14A and 14B, a prolonged plasma half-life followingintravenous administration, particularly for tocopherol succinatecamptothecin, is shown.

The calculated plasma elimination half-life, mean residence time, andclearance of tocopherol succinate camptothecin (SN2300), tocopherolsuccinate 7-ethyl-10-hydroxycamptothecin (SN2310), camptothecin,irinotecan, and topotecan is provided in Table 3.

The term “plasma elimination half-life” refers to the time necessary toreduce the drug concentration in plasma by 50% after equilibrium isreached. The term “elimination rate constant” refers to the fraction ofdrug eliminated per unit of time. With first-order elimination, the rateof elimination is directly proportional to the serum drug concentration.There is a linear relationship between rate of elimination and serumdrug concentration. Although the amount of drug eliminated in afirst-order process changes with concentration, the fraction of a drugeliminated remains constant.

The term “clearance” refers to a measure of the body's ability toeliminate drug and is a hypothetical volume of distribution of drugwhich is cleared per unit time (i.e., mL/min) by any pathway of drugremoval. It is important to clarify that the clearance does not indicatehow much drug is being removed, rather, the volume of biological fluidsuch as blood or plasma that would have to be completely freed of drugto account for the elimination. The term “volume of distribution” refersto a calculated volume of body fluid that would be required to dissolvethe total amount of drug at the same concentration as that found in theblood. It is a proportionality constant relating the amount of drug inthe body to the measured concentration in biological fluid (blood,plasma, serum). TABLE 3 Comparative pharmacokinetic parameters followingintravenous administration in rats of tocopherol succinate camptothecin(SN2300) and tocopherol succinate 7-ethyl-10-hydroxycamptothecin(SN2310) compared to camptothecin, irinotecan, and topotecan. t_(1/2)(hr) MRT Clearance Compound (elimination) (hours) (1/hr/kg) SN2300 29.089.40 0.0081 SN2310 3.49 5.15 0.0067 Camptothecin^(a) 1.7 — 8.81Topotecan^(a) 1.06 — 3.22 Irinotecan^(b) 1.54 1.43 2.22^(a)El-Gizawy SA, Hedaya MA. Cancer Chemother. Pharmacol., 43: 364-370(1999).^(b)Atsumi R, Okazaji O Hakusui H. Biol. Pharm. Bull., 18 (8): 1114-1119(1995).

Table 3 illustrates that the calculated plasma elimination half-life oftocopherol succinate camptothecin (SN2300) and tocopherol succinate7-ethyl-10-hydroxycamptothecin (SN2310) is approximately 30-times and3-times longer than the commercially available analogs, respectively.The higher mean residence time (MRT) and lower clearance rate suggest alonger tumor exposure time to these new derivatives, which may indicatea potential for increased chemotherapeutic effect.

Through lipophilic modification of therapeutic drug compounds, theplasma elimination half-life of the parent therapeutic drug compound canbe increased. The compounds of the invention, by virtue of thelipophilic moiety (e.g., tocopherol moiety), have increased plasmaelimination half-life compared to the parent therapeutic drug. Asillustrated above for tocopherol succinate camptothecin and tocopherolsuccinate 7-ethyl-10-hydroxycamptothecin, the plasma eliminationhalf-life is significantly increased compared to the parent compounds.

Example 19 In Vivo Anti-Tumor Activity of RepresentativeTocopherol-Modified Therapeutic Drug Compounds in Human Tumor Xenographs

In this example, the in vivo anti-tumor activity of representativetocopherol-modified therapeutic drug compounds of the invention,tocopherol succinate camptothecin and tocopherol succinate7-ethyl-10-hydroxycamptothecin, was compared to the anti-tumor effect ofirinotecan.

NCI-H460 Human Tumor Xenograft.

Athymic mice were subcutaneously implanted with a cell suspension (10⁷cells/mouse). When tumors reached an appropriate size, animals wererandomized into groups of eight and intravenously administered thefollowing compounds at a dose of 15 mg of drug compound/kg of bodyweight on a schedule of q1d×5 for two consecutive weeks:

Saline-control group

Irinotecan

SN2300 emulsion

SN2310 emulsion

HT-29 Human Tumor Xenograft.

Athymic mice were subcutaneously implanted with 30-40 mg tumor fragmentsusing 12-gauge trocar needles. A sufficient number of mice wereimplanted with fragments so tumors in narrow weight range (100-200 mg)were selected for the trial on staging day (SD). The animals selectedwith tumors in the proper size range were randomized into six groups of10 animals and intravenously administered the following test compounds:Saline-control group (q1dx5 for 2 weeks) Irinotecan (15 mg/kg, q1dx5 for2 weeks) SN2300 emulsion (15 mg/kg, q1dx5 for 2 weeks) SN2310 emulsion(15 mg/kg, q1dx5 for 2 weeks) SN2300 emulsion (15 mg/kg, q3dx10) SN2310emulsion (15 mg/kg, q3dx10)

In both xenograph studies, animal body weights and tumors were measuredtwice weekly following the initiation of dosing. The tumor measurementswere performed using a caliper (millimeters); the tumor volume wascalculated based on the formula: (Length×width²)/2=Volume (mm³).

The anti-tumor effect of SN2300 and SN2310 emulsions administered toathymic mice implanted with NCI-H460 human tumor cells and HT-29 humantumor cells is graphically represented in FIGS. 15A and 15B,respectively. Although the SN2300 emulsion showed little to noanti-tumor effect in this model, the SN2310 emulsion exhibitedsubstantial anti-tumor effect as compared to both saline control andirinotecan.

The calculated tumor response parameters for the HT-29 xenograft studyis provided in Table 4. Fifty-five days after dose initiation, 30% ofmice in the control group were sacrificed because of tumor size (>4000mm³) and the median tumor size was 3136 mm³. At this same timepoint, 80%of the mice in the SN2310 (q3d×10) group presented no measurable tumor.In addition, the SN2310 (q1d×5) group had a median tumor size of 126 mm³with 40% having no measurable tumor. At the same time, the irinotecangroup showed a median tumor size of 1637 mm³. The results indicate thatthe administration of SN2310 produces significant anti-tumor activity.TABLE 4 Calculated Tumor Response Parameters from the HT-29 XenograftStudy. Tumor Growth Tumor Number of animals Delay Growth with no (T − C)Inhibition measurable Group Schedule (days) (% T/C) tumor on Day 55Saline q1dx5 (2 weeks) — — 0/10 Irinotecan q1dx5 (2 weeks) 14  52 0/10SN2300 q1dx5 (2 weeks)  8.5 79 0/10 SN2300 q3dx10  0 102 0/10 SN2310q1dx5 (2 weeks) >28*  4 4/10 SN2310 q3dx10 >28*  2 8/10*90% of tumors in this group had still not reached maximumpre-determined size.T − C = Median Time for Treatment group (T) and Control group (C) toreach a predetermined size.% T/C = (Treated median tumor weight)/(Control median tumor weight) ×100 (at Day 55).

While the preferred embodiment of the invention has been illustrated anddescribed, it will be appreciated that various changes can be madetherein without departing from the spirit and scope of the invention.

1. Tocopherol succinate paclitaxel.
 2. A method for treating a cellproliferative disease, comprising administering to a subject in needthereof a therapeutically effective amount of tocopherol succinatepaclitaxel.
 3. A method for treating a cell proliferative disease,comprising administering to a subject in need thereof an emulsioncomprising: (a) an oil phase comprising (i) tocopherol succinatepaclitaxel; and (ii) a lipophilic medium; and (b) an aqueous phase. 4.The method of claim 3, wherein the lipophilic medium comprises atocopherol.
 5. A pharmaceutical composition, comprising apharmaceutically acceptable carrier and tocopherol succinate paclitaxel.6. The composition of claim 5, wherein the pharmaceutically acceptablecarrier comprises a lipophilic medium.
 7. The composition of claim 5,wherein the pharmaceutically acceptable carrier comprises a tocopherol.8. Tocopherol succinate docetaxel.
 9. A method for treating a cellproliferative disease, comprising administering to a subject in needthereof a therapeutically effective amount of tocopherol succinatedocetaxel.
 10. A method for treating a cell proliferative disease,comprising administering to a subject in need thereof an emulsioncomprising: (a) an oil phase comprising (i) tocopherol succinatedocetaxel; and (ii) a lipophilic medium; and (b) an aqueous phase. 11.The method of claim 10, wherein the lipophilic medium comprises atocopherol.
 12. A pharmaceutical composition, comprising apharmaceutically acceptable carrier and tocopherol succinate docetaxel.13. The composition of claim 12, wherein the pharmaceutically acceptablecarrier comprises a lipophilic medium.
 14. The composition of claim 12,wherein the pharmaceutically acceptable carrier comprises a tocopherol.